Methods for delivering nucleic acid molecules into cells and assessment thereof

ABSTRACT

Methods for delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of the nucleic acids are provided. The methods are designed for introduction of large nucleic acid molecules, including artificial chromosomes, into cells, and are practiced in vitro and in vivo.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.(attorney dkt. no. 24601-416), to de Jong et al., entitled “METHODS FORDELIVERING NUCLEIC ACID MOLECULES INTO CELLS AND ASSESSMENT THEREOF.”The subject matter thereof is incorporated in its entirety by referencethereto.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of delivering nucleicacid molecules into cells and methods for measuring nucleic aciddelivery into cells and the expression of the nucleic acids therein.

BACKGROUND OF THE INVENTION

[0003] A number of methods of delivering nucleic acid molecules,particularly plasmid DNA and other small fragments of nucleic acid, intocells have been developed. These methods are not ideal for delivery oflarger nucleic acid molecules. Thus, there is a need for methods ofdelivering nucleic acid molecules of increasing size and complexity,such as artificial chromosomes, into cells. Methods are required for usewith in vitro and in vivo procedures such as gene therapy and forproduction of transgenic animals and pants. Furthermore, there is a needfor the ability to rapidly and simply determine and assess theefficiency of delivery of DNA into cells.

[0004] Therefore it is an object herein to provide methods fordelivering nucleic acid molecules, particularly larger molecules,including artificial chromosomes, into cells. Methods for assessingdelivery are also provided.

SUMMARY OF THE INVENTION

[0005] Methods for delivery of large nucleic acid molecules into cellsare provided. The methods, which can be used to deliver nucleic acidmolecules of any size, are suitable for delivery of larger nucleic acidmolecules, such as natural and artificial chromosomes and fragmentsthereof, into cells. The methods are designed for in vitro, ex vivo andin vivo delivery of nucleic acid molecules for applications, including,but limited to, delivery of nucleic acid molecules to cells forcell-based protein production, transgenic protein production and genetherapy. Methods of protein production in cells and in transgenicanimals and plants, methods of introducing nucleic acid into cells toproduce transgenic animals and plants, and methods for ex vivo and invivo gene therapy are also provided.

[0006] Methods provided herein are designed for delivering a largenucleic acid molecule into a cell, but may also be used to deliversmaller molecules. The methods include the steps of exposing the nucleicacid molecule to a first delivery agent, typically an agent thatincreases contact between the nucleic acid molecule and the cell; andexposing the cell to a second delivery agent, which is generallydifferent from the first agent, and is particularly an agent, such asenergy, that enhances permeability of the cell. Selected delivery agentsand combinations thereof are those that result in delivery of thenucleic acid the cell to a greater extent than in absence of the agentor in the presence of one of the agent alone. In all of these methods,if the permeability enhancing agent is energy, such as electroporationor sonoporation, the cell is contacted therewith in the absence of thenucleic acid molecule.

[0007] Also provided are methods in which the cells are contacted with alipid agent, particularly a dendrimer, such as SAINT-2™(1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride,also designated 1-methyl-4-(1 9-cis,cis-hepatritiaconta-9,28-dienyl)pyridinium chloride), simultaneously with or sequentially withapplication of energy. The nucleic acid, which is optionally, althoughpreferably not treated with a delivery agent, is contacted with theso-treated cell.

[0008] The selected delivery methods vary depending on the target cells(cells into which nucleic acid is delivered), the nucleic acidmolecules, and the type(s) of delivery agent(s) selected. Exemplarymethods for delivery of large nucleic acid molecules into cells providedherein include, but are not limited to, methods involving any of thefollowing:

[0009] mixing the nucleic acid molecule with a delivery agent, such as acationic lipid that neutralizes the charge of the nucleic acid, andcontacting the cell with the mixture of nucleic acid and delivery agent;

[0010] contacting a cell with the nucleic acid molecule, and thencontacting the cell with a delivery agent or contacting a cell with adelivery agent then contacting the cell with the nucleic acid molecule;

[0011] contacting a cell in the absence of the nucleic acid moleculewith a delivery agent, applying ultrasound or electrical energy to thecell contacted with the delivery agent, and contacting the cell with thenucleic acid molecule upon the conclusion of the application of theenergy;

[0012] applying ultrasound or electrical energy to a cell, andcontacting the cell, upon conclusion of the application of the energy,with a mixture of the nucleic acid molecule and a delivery agent;

[0013] applying ultrasound or electrical energy to a cell, contactingthe cell with a delivery agent upon conclusion of the application of theenergy and contacting the cell previously contacted with the deliveryagent with the nucleic acid molecule;

[0014] applying ultrasound or electrical energy to a cell and contactingthe cell with the nucleic acid molecule upon conclusion of theapplication of the energy;

[0015] contacting a cell in the absence of the nucleic acid moleculewith a delivery agent, applying ultrasound or electrical energy to thecell contacted with the delivery agent, and contacting the cell with amixture of the nucleic acid and a delivery agent upon the conclusion ofthe application of the energy.

[0016] Although combinations of the above methods may be used, it hasbeen found that any application of energy to the cells must be doneprior to introduction of the nucleic acid molecule.

[0017] The methods provided herein are intended for delivery of largenucleic acid molecules into cells in a variety of environments for avariety of purposes. For example, nucleic acid molecules greater thanabout 0.5, 0.6. 0.7, 0.8, 0.9, 1, 5, 10, 30, 50 and 100 megabase pairsmay be delivered into cells using the methods provided herein. Themethods may be used to deliver the large nucleic acid molecules intocells in vitro or in vivo.

[0018] In in vivo applications of the delivery methods, such as in invivo gene therapy, large nucleic acid molecules may be delivered tocells directly in an animal subject, and in particular human subjects.Reagents can be administered locally or systemically (e.g., in thebloodstream) in the subject. For example, local administration of thenucleic acids, and/or delivery agents, may be into areas such as joints,the skin, tissues, tumors and organs. For systemic administration, thenucleic acid molecules may be targeted to cells or tissues of interest.

[0019] The delivery methods provided herein may also be used to deliverlarge nucleic acid molecules to a target cell in vitro which is thenintroduced into an animal subject, in particular human subjects, such asmay be done, for example, in a method of ex vivo gene therapy. Thus,also provided herein are methods of in vivo and ex vivo gene therapyusing the methods for delivering large nucleic acid molecules into cellsas provided herein.

[0020] In particular embodiments of the methods in which a deliveryagent is used, the delivery agent is a cationic compound. Cationiccompounds include, but are not limited to, a cationic lipid, a cationicpolymer, a mixture of cationic lipids, a mixture of cationic polymers, amixture of a cationic lipid and a cationic polymer and a mixture of acationic lipid and a neutral lipid, polycationic lipids, non-liposomalforming lipids, activated dendrimers, ethanolic cationic lipids,cationic amphiphiles and pyridinium chloride surfactants.

[0021] Included among the nucleic acid molecules that may be deliveredinto cells using the methods provided herein are artificial chromosomes,satellite DNA-based artificial chromosomes (SATACs, herein referred toas ACEs) and natural chromosomes or fragments of any of thesechromosomes.

[0022] The ultrasound energy can be applied as one continuous pulse ortwo or more intermittent pulses. The intermittent pulses of theultrasound energy can be applied for substantially the same length oftime, at substantially the same energy level or can vary in energylevel, the length of time applied, or energy level and the length oftime applied. Ultrasound energy ranges and number of pulses can vary,from methods provided herein, according to the instrument selected andcan be empirically determined. Typically, ultrasound will be applied forabout 30 seconds to about 5 minutes. The power used is a function of thesonorporator used.

[0023] The effects of the ultrasound energy may be enhanced bycon-tacting a cell (in vitro) or administering to a subject (in vivo) acavitation compound prior to the application of ultrasound energy. Thus,the provided methods may include the use of such cavitation compounds.

[0024] When electric fields are employed in the methods provided herein,they are preferably applied to the cells in suspension for about 20 to50 msec, but the timing and voltage is a function of the instrument usedand the particular parameters. The electrical energy can be applied asone to five intermittent pulses. As noted, electrical field ranges andnumber of pulses can vary according to instrument specification and canbe determined empirically.

[0025] Methods are provided for generating transgenic animals,particularly non-human transgenic animals, by delivering large nucleicacid molecules into animal cells, in particular non-human animal cells,using delivery methods provided herein, and exposing the animal cellsinto which the large nucleic acid molecules are delivered to conditionswhereby a transgenic animal develops therefrom.

[0026] The methods for delivering large nucleic acid molecules intocells provided herein may also be used in methods of generatingtransplantable organs and tissues. Exemplary cells for use in methods ofgenerating transgenic animals, particularly non-human transgenicanimals, or transplantable organs include, but are not limited to, anembryonic stem cell, a nuclear transfer donor cell, a stem cell and acell that is capable of the generation of a specific organ. The methodsfor delivering nucleic acid molecules into cells provided herein mayalso be used in methods of generating cellular protein production celllines.

[0027] Further provided are methods for monitoring delivery of nucleicacids into a cell. These methods permit the rapid and accuratemeasurement of nucleic acid transfer into cells, thus allowing forscreening and optimizing the use of various delivery agents andprotocols for delivery of any nucleic acid into any cell type, in vitro,ex vivo or in vivo. Further provided are methods to monitor delivery andexpression of nucleic acids in a cell.

[0028] In embodiments of the methods for monitoring delivery of nucleicacids into a cell, labeled nucleic acid molecules, such as DNA, aredelivered into the cell using the delivery agent(s) as described herein,or using any delivery method known to those of skill in the art. Adetection method, such as flow cytometry, is then used to determine thenumber of cells containing the label as an indication of the ability ofthe delivery method to facilitate or effect delivery of the nucleic acidmolecules. Other detection methods that may be used in place of or inaddition to flow cytometry include, but are not limited to, fluorimetry,cell imaging, fluorescence spectroscopy and other such methods known tothose of skill in the art for such detection and, as needed or desired,for quantitation.

[0029] In an exemplary embodiment of the methods for monitoring andquantifying delivery of nucleic acid molecules, such as DNA, into cells,the nucleic acid molecule is an artificial chromosome labeled with anucleoside or ribonucleoside analog, particularly a thymidine analog,such as iododeoxyuridine (IdU or IdUrd) and bromodeoxyuridine (BrdU),and the delivery agent is a cationic compound, which is used alone or incombination with energy.

[0030] Because of the ease with which numbers of events are collected,the monitoring methods provided herein, particularly those based on flowcytometry techniques, provide a method for collection of nucleic acidmolecule delivery data that is statistically superior to previousmethods of evaluating nucleic acid molecules transfer. The positivevalues are instrument derived and therefore are not susceptible tojudgement errors.

[0031] The monitoring methods provided herein permit the rapid, simpleand accurate detection of delivery of small numbers of nucleic acidmolecules into cells. Such small numbers may be sufficient for purposesof transgenesis, gene therapy, cellular protein production and othergoals of gene transfer. The monitoring methods also make it possible torapidly quantify differences in delivery efficiencies of differingdelivery methods and thus facilitate the development and optimization ofmethods for the delivery of nucleic acid molecules, such as DNA, intocells.

[0032] These methods can also be used to optimize transfectionefficiencies into cells for which no delivery protocol has beenestablished or which are not easily transfected. These methods alsopermit rapid screening of delivery protocols and agents for theirability to enhance or permit delivery of nucleic acid molecules, such asDNA, of any size into a cell.

[0033] Methods are also provided that combine methods of monitoringnucleic acid molecule delivery with methods for monitoring expression ofnucleic acid molecules. It is possible not only to assess the efficiencyof delivery of nucleic acid molecules to cells, but also to monitor thesubsequent expression of the delivered nucleic acid molecules in thesame cell population. Thus, these methods also provide a method for themapping of biological events between nucleic acid molecule delivery andearly gene expression, using marker genes, such as, but are not limitedto, fluorescent proteins, such as red, green or blue fluorescentproteins.

[0034] In a particular embodiment of these combined methods, deliveryand expression of nucleic acid molecules, such as delivery of achromosome and expression of genes encoded thereon, are monitored by IdUlabeling of a nucleic acid molecule that contains sequences encoding agreen fluorescent protein.

[0035] In particular embodiments, the methods of monitoring delivery andexpression of a nucleic acid molecule include the steps of: introducinglabelled nucleic acid molecules that encode a reporter gene into cells;detecting labelled cells as an indication of delivery of the nucleicacid into a cell; and measuring the product of the reporter gene as anindication of DNA expression in the cell, whereby delivery andexpression of nucleic acid molecules in the cell is detected ordetermined. The labelled cells can be detected, for example, by flow d,fluorimetry, cell imaging or fluorescence spectroscopy. The label, forexample, can be iododeoxyuridine (IdU or IdUrd) or bromodeoxyuridine(BrdU), the reporter gene, for example, can be one that encodesfluorescent protein, enzyme, such as a luciferase, or antibody. Thedelivered nucleic acid molecules include, but are not limited to, RNA,including ribozymes, DNA, including naked DNA and chromosomes, plasmids,chromosome fragments, typically containing at least one gene or at least1 Kb, naked DNA, or natural chromosomes. The method is exemplifiedherein by determining delivery and expression of artificial chromosomeexpression systems (Aces). Any types of cells, eukaryotic andprokaryotic, including cell lines, primary cell lines, plant cells, andanimal cells, including stem cells, embryonic cells, and other cellsinto which delivery is contemplated.

DETAILED DESCRIPTION OF THE INVENTION

[0036] A. DEFINITIONS

[0037] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as is commonly understood by one of skillin the art to which this invention belongs. All patents, patentapplications and publications referred to herein are incorporated byreference.

[0038] As used herein, “nucleic acid” refers to a polynucleotidecontaining at least two covalently linked nucleotide or nucleotideanalog subunits. A nucleic acid can be a deoxyribonucleic acid (DNA), aribonucleic acid (RNA), or an analog of DNA or RNA. Nucleotide analogsare commercially available and methods of preparing polynucleotidescontaining such nucleotide analogs are known (Lin et al. (1994) Nucl.Acids Res. 22:5220-5234; Jellinek et al. (1995) Biochemistry34:11363-11372; Pagratis et al. (1997) Nature Biotechnol. 15:68-73). Thenucleic acid can be single-stranded, double-stranded, or a mixturethereof. For purposes herein, unless specified otherwise, the nucleicacid is double-stranded, or it is apparent from the context.

[0039] The term “nucleic acid” refers to single-stranded and/ordouble-stranded polynucleotides, such as deoxyribonucleic acid (DNA) andribonucleic acid (RNA), as well as analogs or derivatives of either RNAor DNA. Also included in the term “nucleic acid” are analogs of nucleicacids such as peptide nucleic acid (PNA), phosphorothioate DNA, andother such analogs and derivatives.

[0040] As used herein, DNA is meant to include all types and sizes ofDNA molecules including cDNA, plasmids and DNA including modifiednucleotides and nucleotide analogs.

[0041] As used herein, nucleotides include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides, such as,but are not limited to, phosphorothioate nucleotides and deazapurinenucleotides and other nucleotide analogs.

[0042] As used herein, the term “large nucleic acid molecules” or “largenucleic acids” refers to a nucleic acid molecule of at least about 0.5megabase pairs (Mbase) in size, greater than 0.5 Mbase, includingnucleic acid molecules at least about 0.6. 0.7, 0.8, 0.9, 1, 5, 10, 30,50 and 100, 200, 300, 500 Mbase in size. Large nucleic acid moleculestypically may be on the order of about 10 to about 450 or more Mbase,and may be of various sizes, such as, for example, from about 250 toabout 400 Mbase, about 150 to about 200 Mbase, about 90 to about 120Mbase, about 60 to about 100 Mbase and about 15 to 50 Mbase.

[0043] Examples of large nucleic acid molecules include, but are notlimited to, natural chromosomes and fragments thereof, especiallymammalian chromosomes and fragments thereof which retain a centromereand telomeres, artificial chromosome expression systems (ACes; alsocalled satellite DNA-based artificial chromosomes (SATACs); see U.S.Pat. Nos. 6,025,155 and 6,077,697), mammalian artificial chromosomes(MACs), plant artificial chromosomes, insect artificial chromosomes,avian artificial chromosomes and minichromosomes (see, e.g., U.S. Pat.Nos. 5,712,134, 5,891,691 and 5,288,625). The large nucleic acidmolecules may include a single copy of a desired nucleic acid fragmentencoding a particular nucleotide sequence, such as a gene of interest,or may carry multiple copies thereof or multiple genes or differentheterologous sequences of nucleotides. For example, ACes can carry 40 oreven more copies of a gene of interest. Large nucleic acid molecules maybe associated with proteins, for example chromosomal proteins, thattypically function to regulate gene expression and/or participate indetermining overall structure.

[0044] As used herein, an artificial chromosome is a nucleic acidmolecule that can stably replicate and segregate alongside endogenouschromosomes in a cell. It has the capacity to act as a gene deliveryvehicle by accommodating and expressing foreign genes contained therein.A mammalian artificial chromosome (MAC) refers to chromosomes that havean active mammalian centromere(s). Plant artificial chromosomes, insectartificial chromosomes and avian artificial chromosomes refer tochromosomes that include plant, insect and avian centromeres,respectively. A human artificial chromosome (HAC) refers to chromosomesthat include human centromeres. For exemplary artificial chromosomes,see, e.g., U.S. Pat. Nos. 6,025,155; 6,077,697; 5,288,625; 5,712,134;5,695,967; 5,869,294; 5,891,691 and 5,721,118 and publishedInternational PCT application Nos, WO 97/40183 and WO 98/08964.

[0045] As used herein, the term “satellite DNA-based artificialchromosome (SATAC)” is interchangable with the term “artificialchromosome expression system (ACes)”. These artificial chromosomes aresubstantially all neutral non-coding sequences (heterochromatin) exceptfor foreign heterologous, typically gene-encoding nucleic acid, that isinterspersed within the heterochromatin for the expression therein (seeU.S. Pat. Nos. 6,025,155 and 6,077,697 and International PCT applicationNo. WO 97/40183). Foreign genes contained in these artificial chromosomeexpression systems can include, but are not limited to, nucleic acidthat encodes traceable marker proteins (reporter genes), such asfluorescent proteins, such as green, blue or red fluorescent proteins(GFP, BFP and RFP, respectively), other reporter genes, such asβ-galactosidase and proteins that confer drug resistance, such as a geneencoding hygromycin-resistance. Other examples of heterologous DNAinclude, but are not limited to, DNA that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and DNA that encodes other types of proteins, such as antibodies.

[0046] As used herein, the terms “heterologous” and “foreign” withreference to nucleic acids, such as DNA and RNA, are usedinterchangeably and refer to nucleic acid that does not occur naturallyas part of a genome or cell in which it is present or which is found ina location(s) and/or in amounts in a genome or cell that differ from thelocation(s) and/or amounts in which it occurs in nature. It is nucleicacid that is not endogenous to the cell and has been exogenouslyintroduced into the cell. Examples of heterologous DNA include, but arenot limited to, DNA that encodes a gene product or gene product(s) ofinterest introduced into cells, for example, for purposes of genetherapy, production of transgenic animals or for production of anencoded protein. Other examples of heterologous DNA include, but are notlimited to, DNA that encodes traceable marker proteins, such as aprotein that confers drug resistance, DNA that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and DNA that encodes other types of proteins, such as antibodies.

[0047] As used herein, “delivery,” which is used interchangeably with“transfection,” refers to the process by which exogenous nucleic acidmolecules are transferred into a cell such that they are located insidethe cell. Delivery of nucleic acids is a distinct process fromexpression of nucleic acids.

[0048] As used herein, “expression” refers to the process by whichnucleic acid is translated into peptides or is transcribed into mRNA andtranslated into peptides, polypeptides or proteins. If the nucleic acidis derived from genomic DNA, expression may, if an appropriateeukaryotic host cell or organism is selected, include splicing of themRNA. For heterologous nucleic acid to be expressed in a host cell, itmust initially be delivered into the cell and then, once in the cell,ultimately reside in the nucleus.

[0049] As used herein, cell recovery refers to a “total cell yield”after a specified time frame, which for purposes herein is twenty-fourhours, and when used with reference to calculation of the clonalfraction

[0050] As used herein, cell recovery time refers to a time frame inorder for a cell to equilibrate to new conditions.

[0051] As used herein, cell survival refers to cell viability after acytotoxic event, such as a delivery procedure.

[0052] As used herein, control plating efficiency (CPE) refers to thefraction of untreated cells, under standard optimal growth conditionsfor the particular cells, that survive a plating procedure. Platingefficiency refers to the fraction of treated cells that survive aplating procedure.

[0053] As used herein, clonal fraction is a measurement of cell recoveryafter delivery of exogenous nucleic acids into cells and the platingefficiency of the cells.

[0054] As used herein, transfer efficiency is the percentage of thetotal number of cells to which nucleic acids are delivered that containdelivered nucleic acid.

[0055] As used herein, transfection efficiency is the percentage of thetotal number of cells to which nucleic acids including a selectablemarker are delivered that survive selection.

[0056] As used herein, index of potential transfection efficiency meansthe theoretical maximum transfection efficiency for a particular celltype under particular conditions, for example particular concentrationsor amounts of particular delivery agents.

[0057] As used herein, the term “cell” is meant to include cells of alltypes, of eukaryotes and prokaryotes, including animals and plants.

[0058] As used herein, “delivery agent” refers to compositions,conditions or physical treatments to which cells and/or nucleic acidsmay be exposed in the process of transferring nucleic acids to cells inorder to facilitate nucleic acid delivery into cells. Delivery agentsinclude compositions, conditions and physical treatments that enhancecontact of nucleic acids with cells and/or increase the permeability ofcells to nucleic acids. In all instances, nucleic acids are not directlytreated with energy, such as sonoporation.

[0059] As used herein, cationic compounds are compounds that have polargroups that are positively charged at or around physiological pH. Thesecompounds facilitate delivery of nucleic acid molecules into cells, itis thought this is achieved by virtue of their ability to neutralize theelectrical charge of nucleic acids. Exemplary cationic compoundsinclude, but are not limited to, cationic lipids or cationic polymers ormixtures thereof, with or without neutral lipids, polycationic lipids,non-liposomal forming lipids, ethanolic cationic lipids and cationicamphiphiles. Contemplated cationic compounds also include activateddendrimers, which are spherical cationic polyamidoamine polymers with adefined spherical architecture of charged amino groups which branch froma central core and which can interact with the negatively chargedphosphate groups of nucleic acids (e.g., starburst dendrimers).

[0060] Cationic compounds for use as delivery agents also includemixtures of cationic compounds that include peptides and proteinfragments. The additional components may be non-covalently or covalentlybound to the cationic compound or otherwise associated with the cationiccompound.

[0061] As used herein, ultrasound energy is meant to include sound waves(for external application) and lithotripter-generated shock waves (forinternal application).

[0062] As used herein, electrical energy is meant to include theapplication of electric fields to cells so as to open pores in membranesfor the delivery of molecules into the cell, e.g., electroporationtechniques.

[0063] As used herein, cavitation compound is meant to include contrastagents that are typically used with ultrasound imaging devices andincludes gas encapsulated and nongaseous agents. These cavitationcompounds enhance the efficiency of energy delivery of acoustic or shockwaves.

[0064] As used herein, “pharmaceutically acceptable” as used hereinrefers to compounds, compositions and dosage forms that are suitable foradministration to the subject without causing excessive toxicity,irritation, allergic response or other undesirable complication.

[0065] As used herein, embryonic stem cells are primitive, immaturecells that are precursors to stem cells.

[0066] As used herein, stem cells are primitive, immature cells that areprecursors to mature, tissue specific cells.

[0067] As used herein, nuclear transfer donor cells are cells that arethe source of nuclei, which are transferred to enucleated oocytes duringthe process of nuclear transfer.

[0068] As used herein, the term “subject” refers to animals, plants,insects, and birds into which the large DNA molecules may be introduced.Included are higher organisms, such as mammals and birds, includinghumans, primates, cattle, pigs, rabbits, goats, sheep, mice, rats,guinea pigs, cats, dogs, horses, chicken and others.

[0069] As used herein, “administering to a subject” is a procedure bywhich one or more delivery agents and/or large nucleic acid molecules,together or separately, are introduced into or applied onto a subjectsuch that target cells which are present in the subject are eventuallycontacted with the agent and/or the large nucleic acid molecules.

[0070] As used herein, “applying to a subject” is a procedure by whichtarget cells present in the subject are eventually contacted with energysuch as ultrasound or electrical energy. Application is by any processby which energy may be applied.

[0071] As used herein, gene therapy involves the transfer or insertionof nucleic acid molecules, and, in particular, large nucleic acidmolecules, into certain cells, which are also referred to as targetcells, to produce specific gene products that are involved in correctingor modulating diseases or disorders. The nucleic acid is introduced intothe selected target cells in a manner such that the nucleic acid isexpressed and a product encoded thereby is produced. Alternatively, thenucleic acid may in some manner mediate expression of DNA that encodes atherapeutic product. This product may be a therapeutic compound, whichis produced in therapeutically effective amounts or at a therapeuticallyuseful time. It may also encode a product, such as a peptide or RNA,that in some manner mediates, directly or indirectly, expression of atherapeutic product. Expression of the nucleic acid by the target cellswithin an organism afflicted with a disease or disorder therebyproviding a way to modulate the disease or disorder. The nucleic acidencoding the therapeutic product may be modified prior to introductioninto the cells of the afflicted host in order to enhance or otherwisealter the product or expression thereof.

[0072] For use in gene therapy, cells can be transfected in vitro,followed by introduction of the transfected cells into the body of asubject. This is often referred to as ex vivo gene therapy.Alternatively, the cells can be transfected directly in vivo within thebody of a subject.

[0073] As used herein, flow cytometry refers to processes that use alaser based instrument capable of analyzing and sorting out cells and orchromosomes based on size and fluorescence.

[0074] As used herein, a reporter gene includes any gene that expressesa detectable gene product, which may be RNA or protein. Preferredreporter genes are those that are readily detectable. Examples ofreporter genes include, but are not limited to nucleic acid encoding afluroescent protein, CAT (chloramphenicol acetyl transferase) (Alton andVapnek (1979), Nature 282: 864-869) luciferase, and other enzymedetection systems, such as beta-galactosidase; firefly luciferase (deWetet al. (1987), Mol. Cell. Biol. 7: 725-737); bacterial luciferase(Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al.(1984), Biochemistry 23: 3663-3667); and alkaline phosphatase (Toh etal. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol.Appl. Gen. 2: 101).

[0075] As used herein, a reporter gene construct is a DNA molecule thatincludes a reporter gene operatively linked to a transcriptional controlsequence. The transcriptional control sequences include the promoter andother optional regulatory regions, such as enhancer sequences, thatmodulate the activity of the promoter, or control sequences thatmodulate the activity or efficiency of the RNA polymerase thatrecognizes the promoter, or control sequences are recognized by effectormolecules, including those that are specifically induced by interactionof an extracellular signal with a cell surface protein. For example,modulation of the activity of the promoter may be effected by alteringthe RNA polymerase binding to the promoter region, or, alternatively, byinterfering with initiation of transcription or elongation of the mRNA.Such sequences are herein collectively referred to as transcriptionalcontrol elements or sequences. In addition, the construct may includesequences of nucleotides that alter translation of the resulting mRNA,thereby altering the amount of reporter gene product.

[0076] As used herein, promoter refers to the region of DNA that isupstream with respect to the direction of transcription of thetranscription initiation site. It includes the RNA polymerase bindingand transcription imitation sites and any other regions, including, butnot limited to repressor or activator protein binding sites, calcium orcAMP responsive sites, and any such sequences of nucleotides known tothose of skill in the art to alter the amount of transcription from thepromoter, either directly or indirectly.

[0077] As used herein, a promoter that is regulated or mediated by theactivity of a cell surface protein is a promoter whose activity changeswhen a cell is exposed to a particular extracellular signal by virtue ofthe presence of cell surface proteins whose activities are affected bythe extracellular protein.

[0078] B. METHODS FOR THE DELIVERY OF DNA INTO CELLS

[0079] A variety of methods for delivering nucleic acids, particularlylarge nucleic acid molecules, such as artificial chromosomes, includingACes (formerly designated SATACs), are provided. The methods generallyinvolve exposing the nucleic acid molecule to an agent that increasescontact between the nucleic acid molecule and the cell, and exposing thecell to a permeability enhancing agent. Each of the methods providedherein requires the use of one or both of these agents, which areapplied in different orders, with the caveat that agents, such asenergy, which increase the permeability of a cell, must be appliedbefore contacting the cell with a nucleic acid.

[0080] In methods provided herein, large nucleic acid molecules aredelivered using agents, including, but not limited to, delivery agentsthat enhance contact between the nucleic acid molecules and the cellsand/or agents and treatments that increase cell permeability. Generallythe nucleic acid molecules are delivered using agents that enhancecontact between the nucleic acid and cells by neutralizing the charge ofthe nucleic acid molecules, and also by using energy to increasepermeability of the cells. The agents may be used individually and invarious combinations and orders of application, with the caveat thatenergy, such as sonoporation and electroporation cannot be applied tocells after the nucleic acid molecule is added thereto.

[0081] The method selected for delivering particular nucleic acidmolecules, such as DNA, to targeted cells can depend on the particularnucleic acid molecule being transferred and the particular recipientcell. Preferred methods for particular nucleic acid molecules, such asDNA, and recipient cells are those that result in the greatest amount ofnucleic acid molecules, such as DNA, transferred into the cell nucleuswith an acceptable degree of cell survival. Suitable methods fordelivery of particular pairings of nucleic acid molecules, such as DNA,and recipient cells may be determined using methods of monitoringnucleic acid molecules, such as DNA, delivery and methods of screeningagents and conditions as provided herein or may be determinedempirically using methods known to those of skill in the art.

[0082] The method selected requires consideration of a number ofparameters, which are discussed in detail below. A method for detectionof delivered nucleic acid is provided. This method, which can be usedfor assessing delivery of any nucleic acid molecule, can be used as arapid screening tool to optimize chromosome transfer conditions.

[0083] In particular, delivery methods may first be assessed for theability to transfer nucleic acid molecules, such as DNA, into cells andto identify methods that provide a sufficient number of viable cellsthat express the transferred nucleic acid molecules, such as DNA. Oncesuch methods are identified, they may be optimized using the deliverymonitoring methods provided herein and then assessed for the ability toprovide for expression of the transferred nucleic acid molecules.

Delivery Agents

[0084] Delivery agents include compositions, conditions and physicaltreatments that enhance contact of nucleic acid molecules, such as DNA,with cells and/or increase the permeability of cells to nucleic acidmolecules, such as DNA. Such agents include, but are not limited to,cationic compounds, peptides, proteins, energy, for example ultrasoundenergy and electric fields, and cavitation compounds.

[0085] Delivery agents for use in the methods provided herein includecompositions, conditions or physical treatments to which cells and/ornucleic acid molecules, such as DNA, may be exposed in the process oftransferring nucleic acid molecules, such as DNA, to cells in order tofacilitate nucleic acid molecules, such as DNA, delivery into cells. Forexample, compounds and chemical compositions, including, but are notlimited to, calcium phosphate, DMSO, glycerol, chloroquine, sodiumbutyrate, polybrene and DEAE-dextran, peptides, proteins, temperature,light, pH, radiation and pressure are all possible delivery agents.

Cationic Compounds

[0086] Cationic compounds for use in the methods provided herein areavailable commercially or can be synthesized by those of skill in theart. Any cationic compound may used for delivery of nucleic acidmolecules, such as DNA, into a particular cell type using the providedmethods. One of skill in the art by using the provided screeningprocedures can readily determine which of the cationic compounds arebest suited for delivery of specific nucleic acid molecules, such asDNA, into a specific target cell type.

(a) Cationic Lipids

[0087] Cationic lipid reagents can be classified into two generalcategories based on the number of positive charges in the lipidheadgroup; either a single positive charge or multiple positive charges,usually up to 5. Cationic lipids are often mixed with neutral lipidsprior to use as delivery agents. Neutral lipids include, but are notlimited to, lecithins; phospho-tidylethanolamine;phosphatidylethanolamines, such as DOPE(dioleoylphosphatidylethanolamine), DPPE(dipalmitoylphosphatidyl-ethanolamine),dipalmiteoylphosphatidylethanolamine, POPE(palmi-toyloleoylphosphatidylethanolamine) anddistearoylphosphatidylethano-lamine; phosphotidylcholine;phosphatidylcholines, such as DOPC (dioleoylphosphidylcholine), DPPC(dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;fatty acid esters; glycerol esters; sphingolipids; cardiolipin;cerebrosides; and ceramides; and mixtures thereof. Neutral lipids alsoinclude cholesterol and other 3βOH-sterols.

[0088] Other lipids contemplated herein, include: phosphatidylglycerol;phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG(dipalmitoylphosphatidylglycerol), and distearoyl-phosphatidylglycerol;phosphatidylserine; phosphatidylserines, such as dioleoyl- ordipalmitoylphosphatidylserine and diphosphatidylglycerols.

[0089] Examples of cationic lipid compounds include, but are not limitedto: Lipofectin (Life Technologies, Inc., Burlington, Ont.)(1:1 (w/w)formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) anddioleoylphosphatidylethanol-amine (DOPE)); LipofectAMINE (LifeTechnologies, Burlington, Ont., see U.S. Pat. No. 5,334,761) (3:1 (w/w)formulation of polycationic lipid 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA) and dioleoyl phosphatidyl-ethanolamine (DOPE)), LipofectAMINEPLUS (Life Technologies, Burlington, Ont. see U.S. Pat. Nos. 5,334,761and 5,736,392; see, also U.S. Pat. No. 6,051,429) (LipofectAmine andPlus reagent), LipofectAMINE 2000 (Life Technologies, Burlington, Ont.;see also International PCT application No. WO 00/27795) (Cationiclipid), Effectene (Qiagen, Inc., Mississauga, Ontario) (Non liposomallipid formulation), Metafectene (Biontex, Munich, Germany) (Polycationiclipid), Eu-fectins (Promega Biosciences, Inc., San Luis Obispo, Calif.)(ethanolic cationic lipids numbers 1 through 12:C₅₂H₁₀₆N₆O₄4CF₃CO₂H,C₈₈H₁₇₈N₈O₄S₂.4CF₃CO₂H, C₄₀H₈₄NO₃P. CF₃CO₂H, C₅₀H₁₀₃N₇O₃.4CF₃CO₂H,C₅₅H₁₁₆N₈O₂.6CF₃CO₂H, C₄₉H₁₀₂N₆O₃.4CF₃CO₂H, C₄₄H₈₉N₅O₃.2CF₃CO₂H,C₁₀₀H₂₀₆N₁₂O₄S₂.8CF₃CO₂H, C₁₆₂H₃₃₀N₂₂O₉.13CF₃CO₂H, C₄₃H₈₈N₄O₂.2CF₃CO₂H,C₄₃H₈₈N₄O₃.2CF₃CO₂H, C₄₁H₇₈NO₈P); Cytofectene (Bio-Rad, Hercules,Calif.) (mixture of a cationic lipid and a neutral lipid), GenePORTER(Gene Therapy Systems Inc., San Diego, Calif.) (formulation of a neutrallipid (Dope) and a cationic lipid) and FuGENE 6 (Roche MolecularBiochemicals, Indianapolis, Ind.) (Multi-component lipid basednon-liposomal reagent).

(b) Non-Lipid Cationic Compounds

[0090] Non-lipid cationic reagents include, but are not limited toSUPERFECT™ (Qiagen, Inc., Mississauga, ON) (Activated dendrimer(cationic polymer:charged amino groups) and CLONfectin™ (Cationicamphiphile N-t-butyl-N′-tetradecyl-3-tetradecyl-aminopropionamidine)(Clontech, Palo Alto, Calif.).

[0091] Pyridinium amphiphiles are double-chained pyridinium compounds,which are essentially nontoxic toward cells and exhibit little cellularpreference for the ability to transfect cells. Examples of a pyridiniumamphiphiles are the pyridinium chloride surfactants such as SAINT-2(1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride)(see, e.g., van der Woude et al. (1997) Proc. Natl. Acad. Sci. U.S.A.94:11 60). The pyridinium chloride surfactants are typically mixed withneutral helper lipid compounds, such as dioleoylphosphatidylethanolamine(DOPE), in a 1:1 molar ratio. Other Saint derivatives of different chainlengths, state of saturation and head groups can be made by those ofskill in the art and are within the scope of the present methods.

Energy

[0092] Delivery agents also include treatment or exposure of the celland/or nucleic acid molecules, but generally the cells, to sources ofenergy, such as sound and electrical energy.

Ultrasound

[0093] For in vitro and in vivo transfection, the ultrasound sourceshould be capable of providing frequency and energy outputs suitable forpromoting transfection. Preferably, the output device can generateultrasound energy in the frequency range of 20 kHz to about 1 MHz. Thepower of the ultrasound energy is preferably in the range from about0.05 w/cm² to 2 w/cm², more preferably from about 0.1 w/cm² to about 1w/cm². The ultrasound can be administered in one continuous pulse or canbe administered as two or more intermittent pulses, which can be thesame or can vary in time and intensity.

[0094] Ultrasound energy can be applied to the body locally orultrasound-based extracorporeal shock wave lithotripsy can be used for“in-depth” application. The ultrasound energy can be applied to the bodyof a subject using various ultrasound devices. In general, ultrasoundcan be administered by direct contact using standard or specially madeultrasound imaging probes or ultrasound needles with or without the useof other medical devices, such as scopes, catheters and surgical tools,or through ultrasound baths with the tissue or organ partially orcompletely surrounded by a fluid medium. The source of ultrasound can beexternal to the subject's body, such as an ultrasound probe applied tothe subject's skin which projects the ultrasound into the subject'sbody, or internal, such as a catheter having an ultrasound transducerwhich is placed inside the subject's body. Suitable ultrasound systemsare known (see, e.g., International PCT application No. WO 99/21584 andU.S. Pat. No. 5,676,151).

[0095] When the cationic compound and nucleic acid molecules, such asDNA, are administered systemically, the ultrasound can be applied to oneor several organs or tissues simultaneously to promote nucleic acidmolecule delivery to multiple areas of the subject's body.Alternatively, the ultrasound can be applied selectively to specificareas or tissues to promote selective uptake of the nucleic acidmolecules, such as DNA.

[0096] The transfection efficiency of the ultrasound can also beenhanced by using contrast reagents, which serve as artificialcavitation nuclei, such as Albunex (Molecular Biosystems, San Diego,Calif.), Imagent (Alliance Pharmaceutical, San Diego, Calif.),Levovist-SHU (Schering AG, Berlin, Germany), Definity (E.I. du Pont deNemour, Wilmington, Del.), STUC (Washington University, St Louis, Mo.)and the introduction of gaseous microbubbles. A contrast reagent can beintroduced locally, such as a joint; introduced systematically, with theenhancement of cavitation efficiency by focusing lithotripter shockwaves at a defined area; or by targeting a contrast reagent to aparticular site and then enhancing cavitation efficiency by focusinglithotripter shock waves.

Electroporation

[0097] Electroporation temporarily opens up pores in a cell's outermembrane by use of pulsed rotating electric fields. Methods andapparatus used for electroporation in vitro and in vivo are well known(see, e.g., U.S. Pat. Nos. 6,027,488, 5,993,434, 5,944,710, 5,507,724,5,501,662, 5,389,069, 5,318,515). Standard protocols may be employed.

[0098] C. TARGET CELLS AND DELIVERY THERETO

In Vitro Delivery

[0099] Cationic compounds and nucleic acid molecules, such as DNA, canbe added to cells in vitro either separately or mixed together and withor without the application of ultrasound or electrical energy, as longas the energy is applied prior to contacting the cells with the nucleicacid molecule.

[0100] In general nucleic acid molecules, such as DNA, mixed withcationic lipids/compounds can be added to cell as described in theEXAMPLES. Parameters important for optimization of the delivery ofnucleic acid molecules, such as DNA, into target cells will be apparentto those of skill in this art. These parameters include, for example,the cationic compound, cationic compound concentration, the nucleic acidmolecules, such as DNA, the concentration of nucleic acid molecules, thecell growth medium, the cell culture conditions, the length of timecells are exposed to the cationic compound, the toxicity of the cationiccompound to the target cell type, and the amount and time of use ofultrasound or electroporation among other parameters. It may benecessary to optimize these parameters for different nucleic acidmolecules, such as DNA, and target cell types. Such optimization isroutine employing the guidance provided herein. In addition, the rapidscreening method can provide direction as to what parameters may need tobe adjusted to optimize delivery (see EXAMPLES). Alteration of cultureconditions, time, reagent concentrations and other parameters, for usewith different combinations of cationic compounds and target cell typesand to optimize delivery, can be empirically determined. If ultrasoundenergy is required to be used to enhance transfection efficiency, it canbe applied as described below and in the EXAMPLES. Electroporation canbe performed as described below or by any suitable protocol known tothose of skill in this art.

[0101] The contacting of cells with cationic compounds and nucleic acidmolecules, such as DNA, in separate and distinct steps can be generallycarried out as described in the EXAMPLES. Those of skill in the art canreadily vary the order of the application of the components to thetarget cell based on the disclosure herein.

Ex Vivo Gene Therapy

[0102] Delivery of nucleic acid molecules, such as DNA, is carried outas described above in in vitro delivery. After selection has beencompleted, cells harboring the nucleic acid molecules, such as DNA, areintroduced into the subject target by a variety of means, includinginjection, such as subcutaneous, intramuscular, intraperitoneal,intravascular and intralymphatic injection. The cells can beadministered with or without the aid of medical devices such asarthroscopes, other scopes or various types of catheters.

In Vivo Gene Therapy

[0103] In one method for delivering of the nucleic acid molecules, suchas DNA, to target cells in the body of a subject in vivo, the cationiccompound is first delivered to the target area (tissue, organ, tumor orjoint). After waiting a suitable amount of time, the target area is thensubjected to ultrasound frequency at a suitable energy level for asuitable time, which will be dependent on the equipment, tissue type anddepth of the target area in the body. Alternatively, electrical energyis delivered to the target area. The nucleic acid molecules, such asDNA, is then delivered to the same area. Optionally, this procedure canbe repeated so that the nucleic acid molecules, such as DNA, can bedelivered via multiple injections over time or multiple administrationsin different areas at the same time.

[0104] The cationic compound mixed together with the nucleic acidmolecules, such as DNA, can be delivered to the target area. The targetarea is then subjected to ultrasound frequency at a suitable energylevel for a suitable time. Depending on the nucleic acid molecules, suchas DNA, the in vivo location, the cationic compound used and othervariables, it may not be necessary to use ultrasound or electroporationto achieve suitable transfer efficiency to cells at the target area.Prior to the application of ultrasound, contrast reagents can bedelivered to the target area to enhance transfer of the nucleic acidmolecules, such as DNA.

[0105] The nucleic acid molecules, such as DNA, can be delivered toorgans or tissues of the body such as skin, muscle, stomach, intestine,lung, bladder, ovary, uterus, liver, kidney, pancreas, brain, heart,spleen, prostate and joints (for example the knee, elbow, shoulder,wrist, hip, finger, and others. Molecules can be delivered to primarycell lines, such as fibroblast, muscle, stomach, intestine, lung,bladder, ovary, uterus, liver, kidney, pancreas, brain, heart, spleen,prostate to mimic in vivo systems.

[0106] The cationic compounds and the nucleic acid molecules, such asDNA, separately or together can be delivered to the target area of thebody by a variety of means, including injection (for example,subcutaneous, intramuscular, intraperitoneal, intravascular andintralymphatic injection), instillation, cannulation, slow infusion,topical application and any other mode of administration. They can beadministered by any suitable mode, including systemically (for exampleby intravenous injection), locally, such as by delivery to a specifictarget area (tissue or area), using, for example, a catheter or bydirect injection. They can be administered with or without the aid ofmedical devices such as arthroscopes, other scopes or various types ofcatheters.

[0107] The cationic compounds can be administered also by coating amedical device, for example, a catheter, such as an angioplasty ballooncatheter, with a cationic compound formulation. Coating may be achieved,for example, by dipping the medical device into a cationic lipidformulation or a mixture of a cationic compound formulation and asuitable solvent, for example, an aqueous-buffer, an aqueous solvent,ethanol, methylene chloride, chloroform and other suitable solvent. Anamount of the formulation will naturally adhere to the surface of thedevice, which is subsequently administered to a subject, as appropriate.Alternatively, a lyophilized mixture of a cationic lipid formulation maybe specifically bound to the surface of the device. Such bindingtechniques are known (see, e.g., Ishihara et al. (1993) Journal ofBiomedical Materials Research 27:1309-1314).

[0108] The cationic compounds and nucleic acid molecules, such as DNA,can be formulated in pharmaceutically acceptable carriers, such assaline or other pharmaceutically acceptable solutions, for delivery invivo. The nucleic acid molecules, such as DNA, and cationic compounds,regardless of the route of administration, are formulated intopharmaceutically acceptable dosage forms by standard methods known tothose of skill in the art.

[0109] For gene therapy, the dosage level of the nucleic acid molecules,such as DNA, may be varied to achieve optimal therapeutic response for aparticular subject. This depends on a variety of factors including modeof administration, activity of the nucleic acid molecules, such as DNA,characteristics of the protein produced, the transfection efficiency ofthe target cells (their ability to take up the nucleic acid molecules,such as DNA), the route of administration, the location of the targetcells and other factors

[0110] The dosage to be administered and the particular mode ofadminis-tration will vary depending upon such factors as the age, weightand the particular animal and region thereof to be treated, theparticular nucleic acid molecule and cationic compound used, thetherapeutic or diagnostic use contemplated, and the form of theformulation, for example, suspension, emulsion, or liposomal, as will bereadily apparent to those skilled in the art. Typically, dosage isadministered at lower levels and increased until the desirabletherapeutic effect is achieved. The amount of cationic compound that isadministered can vary and generally depends upon the amount of nucleicacid molecules, such as DNA, being administered. For example, the weightratio of cationic compound to nucleic acid molecules is preferably fromabout 1:1 to about 15:1, with a weight ratio of about 5:1 to about 1:1being more preferred. Generally, the amount of cationic compound whichis administered will vary from between about 0.1 milligram (mg) to about1 gram (g). By way of general guidance, typically for a bodyweight of 70kg and a composition with about 1 to 10 million chromosomes per ml,single dose ranging from 1 to 20 ml is administered as a single orrepeated dose.

[0111] For localized treatment of diseases, such administration toaffected joints in rheumatoid arthritis, psoriasis and diabetes shouldbe possible, as well as injection into muscle for treatment of diseases,such as hemophilia or other genetic diseases. For other than localtreatment a targeted delivery step is needed.

[0112] D. ASSESSING THE DELIVERY OF NUCLEIC ACID INTO CELLS

[0113] Microscopic and colony formation analysis methods that may beused in evaluating stable nucleic acid molecule delivery rely on manualvisualization or measurement of nucleic acid molecules (e.g., aselectable marker gene) expression, which is a distinct process fromdelivery. Such methods are associated with time delays in obtaining anassessment of the delivery method. Microscopic techniques forvisualizing chromosome or plasmid transfer using bromodeoxyuridine(BrdU) (see. e.g., Pittman et al. J Immunol Methods 103:87-92 (1987))are time consuming, restricted by the large sample size required todetect low levels of transfer and limited by the necessity of manualscoring. Colony-forming transfection analysis may require four-to-sixweeks to generate and evaluate marker-expressing transfection colonies.

[0114] In contrast, methods provided herein are based on rapid,auto-mated, sensitive and accurate analysis procedures, such as flowcytometry, and thus do not involve any time-consuming, laborious anderror-prone steps, such as manual detection of individual transfectedcells by microscopic techniques. The methods make possible the analysisof nucleic acid molecule delivery data within 48 hours aftertransfection. Also, data collected by flow cytometry analysis isstatistically superior due to the ease at which large numbers of events,e.g., nucleic acid molecule transfer, are collected. The positive valuesobtained in these methods are instrument derived and therefore not assusceptible to judgment errors. Thus, these methods provide for greateraccuracy in assessing nucleic acid molecule delivery. In contrast,microscopic analysis is limited by the time involved for scoringpositive events and sample size is restrictive.

[0115] Because the methods of monitoring nucleic acid molecule deliverydetect labeled nucleic acid molecules, such as DNA, and not a reportergene expression product, it is possible to measure absolute values ofnucleic acid molecules transferred, within twenty-four hours, withoutbeing hindered by cell autofluorescence and by the problems ofdifferentiating wild-type cells from cells expressing low levels ofreporter gene products (see, e.g., Ropp et al. (1995) Cytometry21:309-317).

[0116] 1. Factors to consider in addressing delivery of nucleic acids

[0117] Delivery of nucleic acids, including DNA, into cells is a processin which nucleic acids are transferred to the interior of a cell.Methods for the delivery of nucleic acids may be assessed in a varietyof ways, including the following.

a. Transfer Efficiency

[0118] A delivery method may be assessed by determining the percentageof recipient cells in which the nucleic acids, including DNA, is present(i.e., the transfer efficiency). However, when evaluating a deliverymethod for the ultimate goal of generating cells that express thetransferred nucleic acid, there are additional factors beyond merepresence of the nucleic acid in recipient cells that should beconsidered. Included among these additional factors is cell viability.When assessing a proliferating cell population, clonogenicity is themethod of choice to measure viability. When the target cells populationis non-dividing or slow growing, metabolic integrity can be monitored.

b. Clonogenicity

[0119] Clonogenicity represents a measure of the survivability of cellswith respect to a delivery procedure, growth conditions and cellmanipulations (e.g., plating). It is important to assess clonogenicityto determine whether a delivery procedure results in a sufficient numberof viable cells to achieve a desired number of cells containing thetransferred nucleic acid.

[0120] Clonogenicity may be expressed as a clonal fraction. The clonalfraction is an index that is calculated by multiplying two separatefractions and normalizing to a control plating efficiency correctionfactor (CPE). The two separate fractions that are multiplied in thiscalculation are the fraction of cells that survive a delivery procedure(population cell yield) and the fraction of cells that survive a platingprocedure. The calculation is thus as follows:${{Colonal}\quad {Fraction}} = {\frac{\begin{matrix}{\# \quad {viable}\quad {colonies}} \\{{after}\quad {plating}}\end{matrix}}{\# \quad {cells}\quad {plated}} \times \frac{\begin{matrix}{\# \quad {cells}} \\{{post}\text{-}{transfection}}\end{matrix}}{\begin{matrix}{\# \quad {cells}} \\{{transfected} \times {CPE}}\end{matrix}}}$

[0121] The values used in this calculation for the number of cellspost-transfection (i.e., post-delivery) and the number of coloniespost-plating is based on cell or colony numbers at certain times in theprocess. For instance, the value for the number of cellspost-transfection is representative of the number of cells at a timeafter nucleic acid delivery that is sufficient for the delivery processto be completed. This time may be determined empirically. Typically thistime ranges from 4-48 hours and generally is about one day aftertransfection. Likewise, the value of the number of viable coloniespost-plating is representative of the number of colonies at a time afternucleic acid delivery that is sufficient for the non-viable cells to beeliminated and the viable cells to be established as colonies. This timemay be determined empirically. Typically this time ranges from that inwhich the average colony is made up of approximately 50 cells orgenerally is a time at which five cell cycles have passed.

[0122] A correction factor is included to take into account the platingefficiency of control wells, which is the ratio determined by the numberof colonies counted divided by the number cells initially plated(typically 600-1000 cells). For LM(tk-) and V79-4 cells, the value ofthe correction factor typically ranges from about 0.7 to about 1.2 andmay be, for example, 0.9.

[0123] The number of cells plated should remain constant at 1000(simplified plating efficiency assay) done in duplicate, except in thecase where the CPE is below 0.3, then number of cells seeded should beincreased to a range of 5,000-50,000. If the CPE is below 0.1-0.2, thena viable fraction analysis should be considered.

c. Viable Fraction

[0124] If the target cells population is non-dividing or slowly dividingthen reproductive or clonogenicity assays are not relevant. Less directmeasurements of cell viability must be used to measure cell killing thatmonitor metabolic death rather than loss of reproductive capacity. Theseprocedures include, for example: (1) membrane integrity as measured bydye exclusion, (2) inhibition of nucleic acid synthesis as measured byincorporation of nucleic acid precursors, (3) radioactive chromiumrelease, and (4) MTT ASSAY(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). Thesemethods are different from measurements of loss of proliferativecapacity, as they reflect only immediate changes in metabolism, whichcan be reversed or delayed and hence lead to errors in estimation ofcell viability. To minimize these errors, correlation of duplicateprocedures is suggested.

d. Potential Transfection Efficiency (PTE) and Determination of ChromosIndex (CI)

[0125] In assessing a delivery method used to transfer nucleic acids tocells with the goal of expression of the nucleic acids, including DNA,therein, it is desirable to obtain an indication of the theoreticalmaximum percentage of cells that are viable and contain the nucleic acidout of the total number of cells into which nucleic acids weredelivered. This is referred to as the potential transfection efficiencyand may be calculated from existing or historical experimental data setsand is determined as follows:

Potential Transfection Efficiency (PTE)=Transfer Efficiency×(ClonalFraction or Viable Fraction)×correction factor (CF)

[0126] The Chromos Index (C.I.) is an effective and rapid method todetermine the Potential Transfection Efficiency of a proliferatingpopulation by using experimental values of % labeled nucleic acid, suchas ACes, delivery to measure transfer efficiency and clonal fractionmeasured using a simplified clonogenicity assay.

Chromos Index (CI)=% labeled ACes delivery×estimated Clonal fraction×CF

[0127] The values of the transfer efficiency and of the clonal fractionand viable fraction are calculated as described above. The correctionfactor (CF) takes into account sample size, sample time and controlplating efficiency. If all these factors are constant for each variablei.e., sampling time and size then the correction factor will approachthe inverse of the value for the C.P.E., i.e., such that the clonalfraction or transfer efficiency can still approach 100% even with a lowCF, or in other words, if delivery and viability are 100%, then themaximum potential transfection efficiency will equal the platingefficiency of the control cells. The calculation of C.I. allows fordetermination of each variable optimization, with the goal being forparameters, such as transfer efficiency, clonal fraction, and CF toapproach one (or 100%). If sample size or time varies for either clonalfraction or transfer efficiency, then CF represents the extrapolatedvalue based on slope or rate of change. An application of thisassessment is provided in the EXAMPLES.

[0128] A stable transfection efficiency of about 1% is in the range(1-100%) that is considered useful for the introduction of large nucleicacid molecules into target cells. It is possible, using methods providedherein, to predict which delivery methods have to be selected forachieving desired transfection efficiencies without having to growtransfectants for extended times under selective conditions anddetermine numbers of cells surviving selection marker expression. Thisanalysis involves calculation of the Chromos Index (CI) which integratesa “biological” value (the clonal fraction) with a measurement ofchromosomal “uptake” or transfer efficiency (percentage of cellscontaining delivered ACes).

[0129] 2. Labeling of nucleic acid molecules for transfer

[0130] In the methods for monitoring nucleic acid molecule deliveryprovided herein, the nucleic acid molecules, such as DNA, to bedelivered are labeled to allow for detection of the nucleic acidmolecules in recipient cells after transfer into the cells. The nucleicacid molecules may be labeled by incorporation of nucleotide analogs.Any nucleic acid molecule analog that may be detected in a cell may beused in these methods. The analog is either directly detectable, such asby radioactivity, or may be detected upon binding of a detectablemolecule to the analog that specifically recognizes the analog anddistinguishes it from nucleotides that make up the endogenous nucleicacid molecules, such as DNA, within a recipient cell. Analogs that aredirectly detectable have intrinsic properties that allow them to bedetected using standard analytical methods. Analogs may also bedetectable upon binding to a detectable molecule, such as a labeledantibody that binds specifically to the analogs. The label on theantibody is one that may be detected using standard analytical methods.For example, the antibody may be fluorescent and be detectable by flowcytometry or microscopy.

[0131] In particular embodiments of these methods, the nucleic acidmolecules, such as DNA, to be transferred is labeled with thymidineanalogs, such as Iododeoxyuridine (IdUrd) or Bromodeoxyuridine (BrdU).In preferred embodiments, IdUrd is used to label the nucleic acidmolecules, such as DNA, to be transferred. The transferred IdUrd-labelednucleic acid molecules, such as DNA, may be immunologically tagged usingan FITC-conjugated anti-BrdU/IdUrd antibody and quantified by flowcytometry. Thus, the transfer of the labeled nucleic acid molecules,such as DNA, into recipient cells can be detected within hours aftertransfection.

[0132] E. STABILITY OF NUCLEIC ACID MOLECULES TO BE DELIVERED

[0133] It is also of interest to evaluate the stability of the nucleicacid molecule, such as DNA, under the selected delivery conditions. Somedelivery conditions and agents may have adverse effects on nucleic acidmolecule structure. Furthermore, the labeling techniques used in certainmethods of monitoring nucleic acid molecules, such as DNA, delivery mayalso impact nucleic acid molecules, such as DNA, structure and function.

[0134] The effects of delivery conditions on nucleic acid molecules maybe assessed in a variety of ways, including microscopic analysis. In aparticular exemplary analysis of the stability of artificialchromosomes, e.g., ACes, the chromosomes are exposed to the conditionsof interest, e.g., IdU labeling, and analyzed under a fluorescentmicroscope for the ability to remain intact and condensed afterincorporation of nucleotide analogs.

Methods of Monitoring Nucleic Acid Molecule Delivery and Expression

[0135] Methods of monitoring delivery of nucleic acid molecules deliveryprovided herein may also be combined with an assessment of nucleic acidmolecule, such as DNA, expression in recipient cells to provide evenfurther information concerning the overall process of nucleic acidmolecule transfer for purposes of expression.

[0136] For example, to facilitate analysis of nucleic acid molecules,such as DNA, expression, it is desirable to include in the transferrednucleic acid molecules, such as DNA, a reporter gene that encodes areadily detected product. For direct detection, such reporter geneproducts include, but are not limited to green fluorescent proteins(GFP), Red Fluorescent protein (RFP), luciferases, and CAT. For indirectdetection, reporter gene products include, but are not limited, toβ-galactosidase and cell surface markers.

[0137] By using, for example, artificial chromosomes such as ACescontaining a GFP reporter gene, such as, but are not limited to, GFPcoding sequences in combination with labeling of the ACes with DNAanalogs, such as IdU, delivery and expression can be rapidly andaccurately monitored. For example, following the delivery of IdU-labeledGFP gene-containing ACes to target cells by any of the describedmethods, the cells containing the ACes are split into two populations.One population is fixed and stained for IdU and analyzed by flowcytometry to determine percentage delivery. The other population isallowed to go through 4-5 cell divisions (approximately 72 hours), andthe GFP fluorescence is measured as an indication of expression.

[0138] Such studies have revealed that incorporation of the analog labeldoes not affect GFP protein expression, which indicates that the methodsmay be combined to monitor delivery and early expression of the ACes,thus providing more information to rapidly evaluate the efficiency ofdelivery methods. The combined methods can also be used to map thebiological events between the initial stages of delivery and early geneexpression.

[0139] The following examples are included for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLE 1 Preparation of Artificial Chromosomes

[0140] A. GFP Chromosome contained in A9 cell line Plasmids

[0141] Plasmid pIRES-EGFP (see SEQ ID No. 13, plasmid obtained fromClontech, Calif., and is well known, see, e.g., U.S. Pat. Nos.6,034,228, 6,037,133, 5,985,577, 5,976,849, 5,965,396, 5,976,796,5,843,884, 5,962,265, 5,965,396; see, also, U.S. Pat. No. 4,937,190).This plasmid contains the internal ribosome entry site (IRES; Jackson(1990) Trends Biochem. 15:477-483; Jang et al. (1988) J. Virol.62:2636-2643) of the encephalomyocarditis virus (ECMV) between the MCSand the enhanced green fluorescent protein (EGFP) coding region. Thispermits the gene of interest (cloned into the MCS) and the EGFP gene tobe translated from a single bicistronic mRNA transcript. PlasmidplRES2-EGFP is designed for selection, by flow cytometry and othermethods, of transiently transfected mammalian cells that express EGFPand the protein of interest. This vector can also be used to expressEGFP alone or to obtain stably transfected cell lines without drug andclonal selection.

[0142] Enhanced GFP (EGFP) is a mutant of GFP with a 35-fold increase influorescence. This variant has mutations of Ser to Thr at amino acid 65and Phe to Leu at position 64 and is encoded by a gene with optimizedhuman codons (see, e.g., U.S. Pat. No. 6,054,312). EGFP is a red-shiftedvariant of wild-type GFP (Yang et al. (1996) Nucl. Acids Res.24:4592-4593; Haas et al. (1996) Curr. Biol. 6:315-324; Jackson et al.(1990) Trends Biochem. 15:477-483) that has been optimized for brighterfluorescence and higher expression in mammalian cells (excitationmaximum=488 nm; emission maximum=507 nm). EGFP encodes the GFPmut1variant (Jackson (1990) Trends Biochem. 15:477-483) which contains thedouble-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. Thecoding sequence of the EGFP gene contains more than 190 silent basechanges which correspond to human codon-usage preferences (Jang et al.(1988) J. Virol. 62:2636-2643). Sequences flanking EGFP have beenconverted to a Kozak consensus translation initiation site (Huang et al.(1990) Nucleic Acids Res. 18: 937-947) to further increase thetranslation efficiency in eukaryotic cells.

[0143] Plasmid plRES-EGFP was dervied from PlRESneo (orignally calledpCIN4) by replacing the neo gene downstream of the IRES sequence withthe EGFP coding region. The IRES sequence permits translation of twoopen reading frames from one mRNA transcript. The expression cassette ofpIRES-EGFP contains the human cytomegalovirus (CMV) major immediateearly promoter/enhancer followed by a multiple clonging site (MCS), asynthetic intron (IVS; Huang et al. (1990) Nucleic Acids Res. 18:937-947), the EMCV IRES followed by the EGFP coding region and thepolyadenylation signal of bovine growth hormone.

[0144] Location of Features (with reference to SEQ ID No. 13):

[0145] Human cytomegalovirus (CMV) immediate early promoter: 232-820;

[0146] MCS 909-974;

[0147] IVS 974-1269;

[0148] IRES of ECMV 1299-1884;

[0149] Enhanced green fluorescent protein (EGFP) gene 1905-2621;

[0150] fragment containing the bovine polyA signal 2636-2913;

[0151] Col E1 origin of replication 3343-4016; and

[0152] Amplicillin resistance gene 5026-4168

Propagation in E. coli

[0153] Suitable host strains: DH5a, HB101, and other general purposestrains. Single-stranded DNA production requires a host containing an Fplasmid such as JM101 or XL1-Blue.

[0154] Selectable marker: plasmid confers resistance to kanamycin (30μg/ml) to E. coli hosts.

[0155]E. coli replication origin: pUC

[0156] Copy number: ˜500

[0157] Plasmid incompatibility group: pMB1/ColE1

pCHEGFP2

[0158] Plasmid pCHEGFP2 was constructed by deletion of the Nsi1/Smalfragment from plRES-EGFP. Plasmid plRES-EGFP contains the codingsequence for a 2.1 kB Nru 1/Xho fragment of pCHEGFP2 containing the CMVpromoter, synthetic intron, EGFP coding sequence and bovine growthhormone polyadenylation signal. Digestion of plRES-EGFP with Nru 1 andSma 1, yielded a 2.1 kb fragment. Digested DNA was fractionated byagarose gel electrophoresis, the separated band was excised and theneluted from the gel using the Qiaex 11 gel purification system (Qiagen,Mississauga, Ontario).

pFK161

[0159] Cosmid pFK161 was obtained from Dr. Gyula Hadlaczky and containsa 9 kb Notl insert derived from a murine rDNA repeat (see clone 161described in PCT Application Publication No. WO97/40183 by Hadlaczky etal. for a description of this cosmid). This cosmid, referred to as clone161 contains sequence corresponding to nucleotides 10,232-15,000 in SEQID NO. 16. It was produced by inserting fragments of the megachromosome(see, U.S. Pat. No. 6,077,697 and International PCT application No. (WO97/40183); for example, H1D3, which was deposited at the EuropeanCollection of Animal Cell Culture (ECACC) under Accession No. 96040929,is a mouse-hamster hybrid cell line carrying this megachromosome) intoplasmid pWE15 (Stratagene, La Jolla, Calif.) as follows. Half of a 100μl low melting point agarose block (mega-plug) containing isolatedSATACs was digested with Notl overnight at 37° C. Plasmid pWE15 wassimilarly digested with Notl overnight. The mega-plug was then meltedand mixed with the digested plasmid, ligation buffer and T4 ligase.Ligation was conducted at 16° C. overnight. Bacterial DH5α cells weretransformed with the ligation product and transformed cells were platedonto LB/Amp plates. Fifteen to twenty colonies were grown on each platefor a total of 189 colonies. Plasmid DNA was isolated from colonies thatsurvived growth on LB/Amp medium and was analyzed by Southern blothybridization for the presence of DNA that hybridized to a pUC19 probe.This screening methodology assured that all clones, even clones lackingan insert but yet containing the pWE15 plasmid, would be detected.

[0160] Liquid cultures of all 189 transformants were used to generatecosmid minipreps for analysis of restriction sites within the insertDNA. Six of the original 189 cosmid clones contained an insert. Theseclones were designated as follows: 28 (˜9-kb insert), 30 (˜9-kb insert),60 (˜4-kb insert), 113 (˜9-kb insert), 157 (˜9-kb insert) and 161 (˜9-kbinsert). Restriction enzyme analysis indicated that three of the clones(113, 157 and 161) contained the same insert.

[0161] For sequence analysis the insert of cosmid clone no. 161 wassubcloned as follows. To obtain the end fragments of the insert of cloneno. 161, the clone was digested with Notl and BamHl and ligated withNotl/BamHl-digested pBluescript KS (Stratagene, La Jolla, Calif.). Twofragments of the insert of clone no. 161 were obtained: a 0.2-kb and a0.7-kb insert fragment. To subclone the internal fragment of the insertof clone no. 161, the same digest was ligated with BamHl-digested pUC19.Three fragments of the insert of clone no. 161 were obtained: a 0.6-kb,a 1.8-kb and a 4.8-kb insert fragment.

[0162] The insert corresponds to an internal section of the mouseribosomal RNA gene (rDNA) repeat unit between positions 7551-15670 asset forth in GENBANK accession no. X82564, which is provided as SEQ IDNO. 5. The sequence data obtained for the insert of clone no. 161 is setforth in SEQ ID NOS. 6-12. Specifically, the individual subclonescorresponded to the following positions in GENBANK accession no. X82564(i.e., SEQ ID NO. 5) and in SEQ ID NOs. 6-12: Subclone Start End SiteSEQ ID No. in X82564 161k1 7579 7755 NotI, BamHI  6 161m5 7756 8494BamHI  7 161m7 8495 10231 BamHI  8 (shows only sequence corresponding tont. 8495-8950),  9 (shows only sequence corresponding to nt. 9851-10231)161m12 10232 15000 BamHI 10 (shows only sequence corresponding to nt.10232-10600), 11 (shows only sequence corresponding to nt. 14267-15000)161k2 15001 15676 NotI, BamHI 12

[0163] The sequence set forth in SEQ ID NOs. 6-12 diverges in somepositions from the sequence presented in positions 7551-15670 of GENBANKaccession no. X82564. Such divergence may be attributable to randommutations between repeat units of rDNA.

[0164] For use herein, the rDNA insert from the clone was prepared bydigesting the cosmid with Notl and Bgl/ll and was purified as describedabove. Growth and maintenance of bacterial stocks and purification ofplasmids were performed using standard well known methods (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press), and plasmids werepurified from bacterial cultures using Midi- and Maxi-preps Kits(Qiagen, Mississauga, Ontario).

[0165] B. PREPARATION OF THE GFP, MURINE A9 CELL LINE

Cell Culture and Transfection

[0166] The murine A9 cell line was obtained from ATCC and cells werethawed and maintained as described below. Briefly, cells were plated ata density of 2×10⁶ cells per 15 cm tissue culture dish (Falcon, BectonDickinson Labware, Franklin Lakes, N.J.) in growth medium containing of90% DMEM (Canadian Life Technologies Burlington, ON) and 10% FBS (CanSera, Rexdale ON), and were maintained at 37° C., 5% CO₂. Cultures wereroutinely passaged when cells reached 70%-80% confluence. Sub culturingwas carried out as follows: medium was removed by aspiration, 10 ml of1× trypsin-EDTA (Canadian Life Technologies Burlington, ON) wasdispensed onto the cell monolayer and the dish gently swirled todistribute the trypsin-EDTA. Finally, the bulk of the trypsin-EDTA wasremoved by aspiration, and the dish placed at 37° C. for 5 minutes. Toquench the trypsin-EDTA, 10 ml of growth medium was added to the dish,and the single cell suspension was transferred to a 50 ml conical tube.Cell counts were performed using a cell counting apparatus(Beckman-Coulter, Hialeah Fla.). The cells were diluted and re-plated asdescribed above. For cryo-storage, cultures were harvested by treatmentwith trypsin-EDTA, counted and the cell suspension then centrifuged at500×g for 5 minutes in a swinging bucket centrifuge. The cell pellet wasresuspended in freezing medium containing 90% DMEM, 20% FBS and 10% DMSO(Sigma-Aldrich, Oakville, ON) at a density of 1×10⁷ cells/ml. One mlaliquots of the cell suspension were then dispensed into cryo-vials(Nunc, Rochester N.Y.), frozen over night in an isopropanol filledcontainer (NUNC, Rochester N..Y) and placed at −70° C. and thentransferred to the gas phase of a liquid nitrogen freezer for long-termstorage.

[0167] A9 cells were transfected using the Ca₂PO₄ co-precipitationmethod (see, e.g., Graham et al. (1978) Virology 52:456-457; Wigler etal. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376; and (1990)Current Protocols in Molecular Biology, Vol. 1, Wiley Inter-Science,Supplement 14, Unit 9.1.1-9.1.9). One day prior to transfection, A9cells were plated at a density of 2×10⁶ cells per 10 cm dish and 3 hoursbefore transfection the medium was replaced with fresh growth medium.140 μg of the 9 kb rDNA, Notl and 5 μg of the 2.1 kB CMV-EGFP Xhol/Nrulfragments were mixed, co-precipitated and used to prepare the Ca₂PO₄co-precipitate (Calcium Phosphate Transfection System, (Canadian LifeTechnologies Burlington, ON) which was distributed onto 2 10-cm dishesof subconfluent A9 cells. The DNA-Ca₂PO₄ complexes were left on thecells for 18 hours, after which the precipitate was removed byaspiration and cells were subjected to glycerol shock for 1.5 minutes.After glycerol shock, the cell monolayers were gently washed with 2×10ml of dPBS (Canadian Life Technologies Burlington, ON), followed byaddition of 10 ml pre-warmed growth medium. Finally dishes were returnedto the incubator and were maintained at 37° C., 5% CO₂. After 3 hoursrecovery, each dish was passaged onto 3×15 cm tissue dishes

[0168] GFP fluorescence of cultures was monitored visually duringculture using an inverted microscope equipped with epifluorescenceillumination (Axiovert 25; Zeiss, (North York ON) and #41017 Endow GFPfilter set (Chroma Technologies, Brattleboro, Vt.). Enrichment of GFPexpressing populations was carried out as described below.

Enrichment of GFP Expressing Cell Populations by Fluorescence ActivatedCell Sorting

[0169] Cell sorting was carried out using a FACS Vantage flow cytometer(Becton Dickinson Immunocytometry Systems, San Jose, Calif.) equippedwith turbo-sort option and 2 Innova 306 lasers (Coherent, Palo AltoCalif.). For cell sorting a 70 μm nozzle was used. The sheath buffer waschanged to PBS (maintained at 20 p.s.i.). GFP was excited with a 488 nmLaser beam and excitation detected in FL1 using a 500 EFLP filter.Forward and side scattering was adjusted to select for viable cells.Only viable cells were then analyzed for GFP fluorescence. Gatingparameters were adjusted using wild type A9 cells as negative controland GFP CHO cells as positive control.

[0170] For the first round of sorting, A9 cells were harvested 4 dayspost-transfection, resuspended in 10 ml of growth medium and sorted forGFP expressing populations using parameters described above. GFPpositive cells were dispensed into a volume of 5-10 ml of growth mediumsupplemented with 1× penicillin/streptomycin (Canadian Life TechnologiesBurlington, ON) while non-expressing cells were directed to waste. Theexpressing cells were further diluted to 50 ml using the same medium,plated onto 2×15 cm dishes and cultured as described in the previoussection. When the sorted populations reached confluence they werere-sorted to enrich for GFP expressing cells. A total of 4 sequentialsorts were carried out, achieving enrichments of as high as 89% GFPexpressing cells after the final sort. The final GFP expressingpopulations were expanded for cryo-preservation and for fluorescencein-situ hybridization screening (see below). Single cell clones wereestablished from populations of interest by using the flow cytometer todirect GFP expressing single cells to individual wells of 96 wellplates. These were cultured as described above.

Fluorescence In-Situ Hybridization

[0171] Fluorescence In-Situ Hybridization (FISH) screening was carriedout on GFP enriched populations and single cell clones to detectamplification and/or artificial chromosome formation. Preparation ofmetaphase spreads and hybridizations were performed (see, Telenius etal. (1999) Chromosome Res 7:3-7). Probes used include pSAT 1, whichrecognizes the mouse major repeat (see, e.g., Wong et al. (1988) Nucl.Acids Res. 16:11645-11661), pFK161, which hybridizes to the mouserDNA-containing regions and a PCR generated probe against the mouseminor repeat.

[0172] C. Purification of artificial chromosomes by Flow Cytometry andpreparation of DNA from flow sorted chromosomes

[0173] Artificial chromosomes were purified from the host cell by flowcytometry (see de Jong (1999) Cytometry 35:129-133). Briefly,purification was performed on FACS Vantage flow cytometer (BectonDickinson Immunocytometry Systems, San Jose, Calif.) equipped with aTrubo-Sort Option and two Innova 306 lasers (Coherent, Palo Alto,Calif.). The Turbo Sort Option modification include increasing themaximum system pressure from 20 lb/in² to 60 lb/in², increasing the dropdrive frequency from 50,000 drops/s to a maximum of 99,000 drops/s andincreasing the deflection plate voltages from a maximum 6,000 V to 8,000V. Other modifications are made to the instrument to accommodate thehigher pressures. Hoechst 35258 was excited with the primary UV laserbeam, and excitation detected in FL1 by using 420 nm hand-pass filter.Chromomycin A3 was excited by the second laser set at 458 nm andfluorescence detected in FL 4 by using a 475 nm long-pass filter. Bothlasers had an output of 200 mW. Bivariate distributions (1,024×1024channels) were accumulated during each sort. For all chromosome sorts,the sheath pressure was set at 30 lb/in² and a 50 μm diameter nozzle wasinstalled. A drop delay profile was performed every morning and repeatedafter any major plug. Alignment of the instrument was performed daily byusing 3.0 μm diameter Sphero rainbow beads (Spherotech, Libertyville,Ill.). Alignment was considered optimized when a CV of 2.0% or less wasachieved for FL1 and FL4.

[0174] Condensing agents (hexylene glycol, spermine and spermidine) wereadded to the sheath buffer to maintain condensed chromosomes aftersorting. The sheath buffer contains 15 nM Tris HCl, 0.1 mM EDTA, 20 mMNaCl, 1% hexylene glycol, 100 mM glycine, 20 μM spermine and 50 μMspermidine. The sorted chromosomes were collected in 1.5 ml screw-cappedEppendorf tubes at 4° C. at a concentration of approximately 1×10⁶chromosomes/ml, which were then stored at 4° C.

[0175] For preparation of purified genomic DNA, sorted chromosomesamples were brought to 0.5% SDS, 50 mm EDTA and 100 μg/ml Proteinase K,then incubated for 18 hours at 50° C. 1 μl of a 20 mg/ml glycogensolution (Boehringer Mannheim) was added to each sample, followed byextraction with an equal volume of Phenol: Chloroform: Isoamyl Alcohol(25:24:1). After centrifugation at 21,000 Xg for 10 min, the aqueousphases were transferred to fresh microfuge tubes and were re-extractedas above. 0.2 volumes of 10 M NH₄OAC, 1 μl of 20 mg/ml glycogen and 1volume of iso-propanol were added to the twice extracted aqueous phaseswhich were then vortexed and centrifuged for 15 minutes at 30,000×g (atroom temperautre). Pellets were washed with 200 μl of 70% ethanol andre-centrifuged as above. The washed pellets were air-dried thenresuspended in 5mM Tris-Cl, pH 8.0 at 0.5-2×10⁶ chromosomeequivalents/μl.

[0176] PCR was carried out on DNA prepared from sorted chromosomesamples essentially as described (see, Co et al. (2000) ChromosomeResearch 8:183-191) using primers sets specific for EGFP and RAPSYN.Briefly, 50 μl PCR reactions were carried out on genomic DNA equivalentto 10,000 or 1000 chromosomes in a solution containing 10 mM Tris-CI, pH8.3, 50 mM KCI, 200 μM dNTPs, 500 nM of forward and reverse primers, 1.5mM MgCl₂, 1.25 units Taq polymerase (Ampli-Taq, Perkin-Elmer Cetus,Calif.). Separate reactions were carried out for each primer set. Thereaction conditions were as follows: one cycle of 10 min. at 95° C.,then 35 cycles of 1 min. 94° C., 1 min. 55° C., 1 min 72° C., andfinally one cycle of 10 min at 72° C. After completion the samples wereheld at 4° C. until analyzed by agarose gel electrophoresis using thefollowing primers (SEQ ID Nos. 1-4, respectively):

[0177] EGFP forward primer 5′-cgtccaggagcgcaccatcttctt-3′;

[0178] EGFP reverse primer 3′-atcgcgcttctcgttggggtcttt-3′;

[0179] RAPSYN forward primer 5′-aggactgggtggcttccaactcccagacac-3′; and

[0180] RAPSYN reverse primer 5′-agcttctcattgctgcgcgccaggttcagg-3′.

[0181] All primers were obtained from Canadian Life Technologies,Burlington, ON.

EXAMPLE 2 Preparation of Cationic Vesicles

[0182] Vesicles were prepared at a lipid concentration of 700 nmoles/mllipid (cationic lipid/DOPE 1:1) as follows. In a glass tube (10 ml) 350nmoles cationic lipid (SAINT-2) was mixed with 350 nmolesdioleyl-phosphoethanolamine (DOPE), both solubilized in an organicsolvent (Chloroform, Methanol or Chloroform/Methanol 1:1, v/v).Diphosphatidyl-ethanolamine (DOPE; Avanti Polar Lipids, Alabaster, Ala.)forms inverse hexagonal phases in a membrane and weakens the membrane.Other effectors that may be used are cis-unsaturatedphosphoethanolamines, cis-unsaturated fatty acids, cholesterol.Cis-unsaturated phosphatidylcholines are less effective.

[0183] The solvent was evaporated under a stream of nitrogen (15 min/250μl solvent at room temperature). The remaining solvent was removedtotally by drying the lipid for 15 min in an desiccator under highvacuum from a vacuum pump. To the dried mixture was added 1 ml ultrapurewater. This was vortexed vigorously for about 5 min. The resultingsolution was sonicated in an ultrasonication bath (Laboratory SuppliesInc. N.Y.) until a clear solution was obtained. The resulting suspensioncontained a population of unilamellar vesicles with a size distributionbetween 50 to 100 nm.

EXAMPLE 3 Preparation of Cationic Vesicles via Alcoholic Injection

[0184] In a glass tube (10 ml) 350 nmoles cationic lipid (Saint-2) wasmixed with 350 nmoles DOPE, both solubilized in an organic solvent(chloroform, methanol or chloroform/methanol 1/1). The solvent wasevaporated under a stream of nitrogen (15 min/250 μl solvent at roomtemperature). The remaining solvent was removed totally by drying thelipid for 15 min under high vacuum. This was then reconstituted in 100μl pure ethanol.

EXAMPLE 4 Transfection of Beta ACes into V79-4 Cell Line TransfectionProcedure for Various Transfection Agents

[0185] All compounds were tested in a Chinese Hamster lung fibroblastline (V79-4, ATCC number CCL-39). Approximately 17 hours (2 celldoublings) prior to transfection, exponentially growing cells weretrypsinized and plated at 250,000 cells per well into a 6 well petridish with Dulbecco's Modified Eagle Medium (Life Technologies,Burlington, ON) and supplemented with 10% FBS (Can Sera, Rexdale ON)).At the time of transfection, the number of cells per well was estimatedto be approximately 1 million. For transfection, each individualmanufacturer's protocol for complexing to naked DNA was followed, withthe exception that the amount of transfection agent used was varied, toreflect the different amount and type of DNA present, as well as thedifferent ionic strength of the complexing. One million ACes (in avolume of 800 μl) were typically combined with the transfection agent ina wide range of concentrations (between 5 times and 100 times the lowestmanufacturers suggested concentration). The ACes/transfection mixturewas allowed to complex for the time recommended by the manufacturer, involumes ranging from 0.8 ml to 1.9 ml; some manufacturers recommendadding media to the complexing reaction. The complexed mixture was thenapplied to the recipient cells and transfection allowed to proceedaccording to the manufacturer's protocol. Details on the variousconditions used with different agents are presented in Table 1.

Transfection Procedure for Superfect Agent

[0186] Superfect was tested in a Chinese Hamster lung fibroblast line(V79-4, ATCC number CCL-39). Approximately 17 hours (2 cell doublings)prior to transfection, exponentially growing cells were trypsinized andplated at 250,000 cells per well into a 6 well petri dish withDulbecco's Modified Eagle Medium (Life Technologies, Burlington, ON) andsupplemented with 10% FBS (Can Sera, Rexdale ON). One million ACes in800 μl of sort buffer was complexed to 10 μl of Superfect reagent.Complex was incubated at room temperature for 10 minutes. At the time oftransfection, the number of cells per well was estimated to beapproximately 1 million. Media was removed from wells and 600 μl of DMEMand 10% FBS was added. Superfect:ACes complex was added to the wellsdrop-wise and allowed to incubate for 3 hours at 37° C. Afterincubation, transfected cells were trypsinized and transferred to 15 cmdishes with 25 ml DMEM and 10% FBS and allowed to attach for 24 hours.After 24 hours, selection medium containing of 0.7 mg/ml hygromycin Bwas added to each well. The selection medium was changed every 2-3 days.After 10-12 days colonies were screened for Beta-galactosidaseexpression and/or FISHed for detection of intact chromosome.

Example of Application of the Determination of the Chromos Index

[0187] Approximately 1×10⁶ V79-4 cells were transfected with 1×10⁶IdUrd-labeled ACes complexed with a delivery agent (i.e., LipofectaminePLUS and Lipofectamine or Superfect). The transfected cells were thenfixed in ethanol. Fixed cells were denatured and exposed toFITC-conjugated antibody that specifically binds to BrdU/IdUrd-labelednucleic acids.

[0188] The percentage of transfected cells containing IdUrd-labeled ACeswas determined using flow cytometry and collecting FITC fluorescence.Data were accumulated to form bivariate channel distribution showingforward scatter versus green fluorescence (IdUrd-FITC). The fluorescencelevel at which cells were determined to be positive was established byvisual inspection of the histogram of negative control cells such thatthe gate for the negative cells was set such that 1% appeared in thepositive region.

[0189] The number of cells recovered at 24 hours post-transfection wasdetermined by counting an aliquot using a Coulter Counter. To determinethe control plating efficiency of a recipient cell line, the untreatedcells were plated at 600-1000 cells per 10 cm petri dish in growthmedium and left stationary in a 5% CO₂ incubator at 37° C. forapproximately five cell cycles or until average colony was made up of 50cells. At this point the number of viable colonies was determined. Thetreated cells were seeded at 1000 cells if the CPE is above 0.1-0.2. Ifthe CPE is low then the seeding density is increased to 5,000-50,000cells per dish.

EXAMPLE 5 Ultrasound Mediated Transfection of LMTK(-) Cells withLipofectamine

[0190] LM(tk-) cells were grown at 37° C., 5% CO₂, in DMEM with 4500mg/L D-glucose, L-glutamine, pyridoxine hydrochloride and 10% FetalBovine Serum. The corner wells of a 12-well dish were seeded with200,000 cells per well (this is to ensure no interference from theultrasound waves from other wells) 24 hours before use.

[0191] The GFP chromosomes were counted to verify approximately 1×10⁶ACes per ml. The chromosomes were resuspended in the tube by flicking.Ten μl of chromosome suspension was removed and mixed with an equalvolume of 30 mg/ml PI (propidium iodide) stain. Eight μl of the stainedchromosomes was loaded onto a Petroff Hausser counting chamber and thechromosomes were counted.

[0192] The medium was removed from the cells, and the cells were washedtwice with HBSS (without phenol red, Gibco BRL) warmed to 37° C. 500 μlof the warmed HBSS was added to each well of cells (1 μl) LipofectAMINE(Gibco BRL) was added to each well. The plates were then sealed withparafilm tape and shaken gently at 20 rpm at room temperature for 30minutes (Stagger plates—10 minutes for ease of handling).

[0193] After incubation Ultrasound gel (Other-Sonic Generic Ultra soundtransmission gel, Pharmaceutical Innovations, Inc., Newark, N.J.) wasapplied to the 2.5 cm sonoporator head. Ultrasound was applied with anImaRX Sonoporator 100 at an output energy of 2.0 Watt/cm2, for 60seconds, through the bottom of the plate of cells. After ultrasound ofthe well one chromosome per seeded cell (2×10⁵) or 200 μl GFP ACes insheath buffer (15 nM Tris HCl, 0.1 mM EDTA, 20 mM NaCl, 1% hexyleneglycol, 100 mM glycine, 20 μM spermine and 50 μM spermidine) are addedimmediately to the well. (Repeat until all samples on the platerequiring ultrasound have been treated). The plate was then sealed oncemore with parafilm tape and shaken gently (20 rpm) for 1 hour at roomtemperature.

[0194] After the incubation 1 ml (DMEM with 4500 mg/L D-glucose,L-glutamine and pyridoxine hydrochloride, 10% Fetal Bovine Serum, and a1× solution of penicillin and streptomycin from a 10000 units/mlpenicillin and 10000 mg/ml Streptomycin, 100× stock solution) was addedto each well and the cells were incubated 18-24 hours at 37° C.

[0195] The cells in the plates were then washed with antibioticcontaining medium and 2 ml of medium was placed in each well. The cellscontinued to be incubated at 37° C. with 5% CO₂ until 48 hours aftertransfection/sonoporation. The cells were then trypsinised andresuspended at a concentration of 1×10⁶ in DMEM to be analyzed by flowcytometry.

[0196] Results: Flow analysis was performed on a FACS Vantage (BDIS, SanJose, Calif.) equipped with a turbo-sort option and two Inova 305 lasers(Coherent, Palo Alto, Calif.). The GFP signal excitation is at 488 nmand the emission detected in FL1 using a 500 nm long pass filter.Analysis of the transfected cells generated populations of GFP positivecells ranging from 13-27%. Non-sonoporated control value was 5%.

EXAMPLE 6 Ultrasound Mediated Transfection with Saint-2

[0197] A. Ultrasound mediated transfection of CHO-KI cells with Saint-2

[0198] CHO-KI cells were grown at 37°, 5% CO₂, in CHO-S-SFM 2 Medium,(Gibco BRL, Paisley, UK). Between 2×10⁵ and 5×10⁵ cells were plated ontosterile glass slides in a 12 well plate 24 h before usage.

[0199] Transfection of the cells was performed as follows. The mediumwas removed from the cells, and the cells were washed twice with HBSS(Hanks balanced salt solution without Phenol Red (Gibco BRL, UK)) at 37°C. Then 500 μl HBSS at 37° C. was added per well, followed by 10 μl ofthe freshly prepared vesicle solution (prepared in Example 2) to yield afinal concentration of 23.3 nmole/ml.

[0200] Alternatively, the medium was removed from the cells, and thecells were washed twice with HBSS. 500 μl HBSS/lipid solution at 37° C.was added to each well. The HBSS/lipid solution was prepared by adding 1μl ethanolic lipid solution (prepared as described above) to 500 μl HBSSunder vigorous vortexing. The plates were then sealed with parafilm tapeand shaken gently at room temperature for 30 min. After incubation,ultrasound was applied at an output energy of 0.5 Watt/cm² for 60 secthrough the bottom of the plate to the cells. The ultrasound wasmediated by an ultrasound gel (Aquasonic 100, Parker, N.J.) betweentransducer and plate. The ultrasound was applied with an ImaRxSonoporator 100. Immediately after applying ultrasound one GFPchromosome per seeded cell (2×10⁵−5×10⁵) (prepared in Example 1) wasadded. The plate was then sealed again and shaken gently for 1 h at roomtemperature. After the incubation 1 ml medium (CHO-S-SFM 2 with 10%Fetal Calf Serum, 10000 μg/ml Penicillin and 10000 μg/ml StreptomycinGibco BRL, Paisley, UK) was added to each well and the cells wereincubated for 24 h at 37° C. The cells were then washed with medium and1 ml medium was added and the cells were incubated at 37° for another 24h. Detection of expressed genes was then assayed by microscopy ordetection of the transferred chromosome by FISH analysis. The negativecontrol was performed in the same way, but with no chromosomes added tothe cells.

Results

[0201] After transfection, using visual inspection, 30% of the cellsremained on the glass slide of which 10% were positive for greenfluorescent protein expression after 48 hours (3% of originalpopulation). After culturing for two weeks, FISH was performed on thecells and 1.4% of the cells contained an intact artificial chromosome.

[0202] B. Ultrasound mediated transfection of Hep-G2 cells with Saint-2

[0203] Hep-G2 cells were grown at 37° C., 5% CO₂, in DMEM with 4500 mg/lGlucose, with Pyridoxine/HCL, 10% Fetal Calf Serum, 10000 μg/mlStreptomycin and 1000 μg/ml Penicillin. Between 2×10⁵ and 5×10⁵ cellswere plated onto sterile glass slides in a 12 wells plate 24 hoursbefore usage.

[0204] Cells were transfected with GFP chromosomes using the procedureof Example 6A except that the CHO-KI medium was replaced with Hep-G2medium.

Results

[0205] After transfection, 30% of the cells remained on the glass slide.80% of these cells were positive for green fluorescent proteinexpression.

[0206] C. Ultrasound mediated transfection of A9 cells with Saint-2

[0207] A9 cells were grown at 37° C., 5% CO₂, in DMEM with 4500 mg/lGlucose, with Pyridoxine/HCL, 10% Fetal Calf Serum, 10000 μg/mlStreptomycin and 10000 μg/ml Penicillin (GIBCO BRL, Paisley, UK).Between 2×10⁵ and 5×10⁵ cells were plated onto sterile glass slides in a12 well plate 24 h before usage.

[0208] Cells were transfected with GFP chromosomes using the procedureof Example 6A except that CHO-KI medium was replaced with A9 medium.

Results

[0209] After transfection, 30% of the cells remained on the glass ofwhich 50% were positive for green fluorescent protein expression.

EXAMPLE 7

[0210] A flow cytometry technique for measuring delivery of artificialchromosomes

[0211] Production cells lines (see Example 1) were grown in MEM medium(Gibco BRL) with 10% fetal calf serum (Can Sera, Rexdale ON) with 0.168μg/ml hygromycin B (Calbiochem, San Diego, Calif.). Iododeoxyurine orBromodeoxyurdine were added directly to culture medium of the productioncell line (CHO E42019) in the exponential phase of growth. StockIododeoxyurdine made in tris base pH 10. Bromodeoxyurdine stocks in PBS.Final concentrations of 0.05-1 μM for continuous label of 20-24 hours of5-50 μM with 15 minute pulse. After 24 hours, exponentially growingcells blocked in mitosis with colchicine (1.0 μg/ml for 7 hours beforeharvest. Chromosomes were then isolated and stained with Hoechst 33258(2.5 μg/ml) and chromomycin A3 (50 μg/ml). Purification of artificialchromosomes was performed using a FACS Vantage flow cytometer (BectonDickinson Immunocytometry systems, San Jose, Calif.). Chromomycin A3 wasexcited with the primary laser set at 457 nm, with emission detectedusing 475 nm long pass filter. Hoechst was excited by the secondary UVlaser and emission detected using a 420/44 nm band-pass filter. Bothlasers had an output of 150 mW. Bivariate distribution showing cellkaryotype was accumulated from each sort. ACes were gated from otherchromosomes and sorted. Condensing agents (hexylene glycol, spermine,and spermidine) were added to the sheath buffer to maintain condensedintact chromosome after sorting. IdU labeling index of sortedchromosomes was determined microscopically. Aliquot (2-10 μl) of sortedchromosomes fixed in 0.2% formaldehyde solution for 5 minutes beforebeing dried on clean microscopic slide. Microscope sample fixed with 70%ethanol. Air-dried slide denatured in coplin jar with 2NHCL for 30minutes at room temperature and washed 2-3 times with PBS. Non specificbinding blocked with PBS and 4% BSA or serum for minimum of 10 minutes.A ⅕ dilution of FITC conjugated IdU/BrdU antibody (Becton Dickinson)with a final volume of 60-100 μl applied to slide. Plastic strips, Durraseal (Diversified Biotech, Boston, Mass.) overlaid on slides, and slideskept in dark at 4%C in humidified covered box for 8-24 hours. DAPI(Sigma) 1 μg/ml in Vectorshield used as counterstain. Fluorescencedetected using Zeiss axioplan 2 microscope equipped for epiflorescence.Minimum of 100 chromosomes scored for determining % labeled. Unlabeledchromosomes used as negative control.

[0212] The day before the transfection, trypsinize V79-4 (ChineseHamster Lung fibroblast) cells and plate at 250,000 into a 6 well petridish in 4 mls DMEM (Dulbecco's Modified Eagle Medium, Life Technologies)and 10% FBS (Can Sera Rexdale ON). The protocol modified for use with LM(tk-) cell line by plating 500,000 cells. Lipid or dendrimer reagent wasadded to 1×10⁶ ACes sorted in ˜800 μl sort buffer. Exemplary protocolvariations are set forth in Table 1. Chromosome and transfection agentswere mixed gently. Complexes added to cells drop-wise and plate swirledto mix. Plates kept at 37° C. in a 5% CO₂ incubator for specifiedtransfection time. The volume in a well was then made up to 4-5 ml withDMEM and 10% FBS. Recipient cells left for 24 hours at 37° C. in a 5%CO₂ incubator. Trypsinize transfected cells. Samples to be analyzed forIdU labeled chromosome delivery are fixed in cold 70% ethanol and storedat −20° C., tp be ready for IdU antibody staining. Samples to be grownfor colony selection are counted and then transferred to 10-cm dishes atdensities of 10,000 and 100,000 cells in duplicate with remaining cellsput in a 15 cm dish. After 24 hours, selection medium containing of DMEMand 10% FBS with 0.7 mg/ml hygromycin B, #400051 (Calbiochem San Diego,Calif.) is added. Selection medium is changed every 2-3 days. Thisconcentration of hygromycin B kills the wild type cells after selectionfor 7 days. At 10-14 days colonies expanded and then screened by FISHfor intact chromosome transfer and assayed for beta galactosidaseexpression. TABLE 1 Delivery Transfection Protocols Medium (ml) PreComplexing added to wells treatment time Added to before TransfectionAgent Dilution Stock of ACes (minutes) complexes complexes time (hours)CLONFECTIN 2-8 μg in 20 1.8 ml of 4 NaCl-HEPES serum free CYTOFECTENE10-20 200 μl of 50% 24  FBS plus DMEM ENHANCER + Enhancer 5 10 1.2 3EFFECTENE minutes (1:5 ratio) EU-FECTIN-1 to  5-10 6 11 FUGENE 6 0.5-6μl to 15-45 4 final volume of 100 μl in serum free medium GENEPORTER 22.5 μl added  2-10 2-4 to 150 μl of serum free medium LIPOFECTAMINE 15 3LIPOFECTAMINE 20 2.5 5 2000 METAFECTENE diluted into 15-45 0.8 6 60 μlserum free medium PLUS + PLUS and 200 15 3 LIPOFECTAMINE μl of DMEM (1:1and 3:2 for 15 minutes ratio) SUPERFECT 10 0.6 3

[0213] IDU ANTIBODY LABELING

[0214] A standard BrdU staining flow cytometry protocol (Gratzer et al.Cytometry (1981);6:385-393) used except with some modifications atneutralization step, the presence of detergent during denaturation andthe composition of blocking buffer. Between each step samples arecentrifuged at 300 g for 7-10 minutes and supernatant removed. Samplesof 1-2 million cells are fixed in 70% cold ethanol. Cells are thendenatured in 1-2 ml of 2N HCL plus 0.5% triton X for 30 minutes at roomtemperature. Sample undergoes 3-4 washes with cold DMEM until indictoris neutral. Final wash with cold DMEM plus 5% FBS.Blocking/permeabilization buffer containing PBS, 0.1% triton X and 4%FBS is added for 10-15 minutes before pelleting sample bycentrifugation. Add 20 μl of IdU/BrdU FITC conjugated B44 clone antibody(Becton Dickinson Immunocytometry Systems, San Jose, Calif.) to pelletand leave for 2 hours at room temperature in the dark with agitationevery 30 minutes. Wash cells with block/permeabilization buffer andresuspend in PBS for flow analysis.

FLOW CYTOMETRY DETECTION OF FLUORESCENT IDUrdD LABELED ACes

[0215] Percentage of transfected cells containing IdU labeled ACes wasdetermined using a flow cytometry with an argon laser turned to 488 nmat 400 mW. FITC fluorescence was collected through a standard FITC530/30-nm band pass filter. Cell populations were gated on the basis ofside scatter versus forward scatter to exclude debris and doublets. Datawas accumulated (15,000 events) to form bivariate channel distributionshowing forward scatter versus green fluorescence (IdU-FITC). Thefluorescence level at which cells were determined to be positive wasestablished by visual inspection of the histogram of negative controlcells, such that approximately 1% appeared in the positive region.

Results:

[0216] The transfection delivery results of IdU labeled ACes are setforth in Table 2. TABLE 2 DOSE DELIVERY Microliters agent % IdU positiveCOMPOUND added per 1 (24 hours) CLONFECTIN 6 0.61 CYTOFECTENE 8 14.67ENHANCER + EFFECTENE 1.6, 10 17.08 (1:5) EU-FECTIN-1 10 4.57 EU-FECTIN-25 0.14 EU-FECTIN-3 10 0.69 EU-FECTIN-4 10 0.24 EU-FECTIN-5 10 0.41EU-FECTIN-6 10 0.46 EU-FECTIN-7 10 1.21 EU-FECTIN-8 10 1.58 EU-FECTIN-910 0.6 EU-FECTIN-10 10 0.77 EU-FECTIN-11 5 1 FUGENE 8 0.49 GENEPORTER 522.12 LIPOFECTAMINE 25 17.81 LIPOFECTAMINE 2000 30 10.96 PLUS +LIPOFECTAMINE  12, 12 12.2 (1:1) PLUS + LIPOFECTAMINE  24, 16 26.97(3:2) METAFECTENE 10 14.14 SUPERFECT 2 27.67

[0217] Since modifications will be apparent to those of skill in thisart, it is intended that this invention be limited only by the scope ofthe appended claims.

1 13 1 24 DNA Artificial Sequence Primer 1 cgtccaggag cgcaccatct tctt 242 24 DNA Artificial Sequence Primer 2 atcgcgcttc tcgttggggt cttt 24 3 30DNA Artificial Sequence Primer 3 aggactgggt ggcttccaac tcccagacac 30 430 DNA Artificial Sequence Primer 4 agcttctcat tgctgcgcgc caggttcagg 305 22118 DNA Mus musculus 5 gaattcccct atccctaatc cagattggtg gaataacttggtatagatgt ttgtgcatta 60 aaaaccctgt aggatcttca ctctaggtca ctgttcagcactggaacctg aattgtggcc 120 ctgagtgata ggtcctggga catatgcagt tctgcacagacagacagaca gacagacaga 180 cagacagaca gacagacgtt acaaacaaac acgttgagccgtgtgccaac acacacacaa 240 acaccactct ggccataatt attgaggacg ttgatttattattctgtgtt tgtgagtctg 300 tctgtctgtc tgtctgtctg tctgtctgtc tatcaaaccaaaagaaacca aacaattatg 360 cctgcctgcc tgcctgcctg cctacacaga gaaatgatttcttcaatcaa tctaaaacga 420 cctcctaagt ttgccttttt tctctttctt tatctttttcttttttcttt tcttcttcct 480 tccttccttc cttccttcct tccttccttt ctttctttctttctttcttt cttactttct 540 ttctttcctt cttacattta ttcttttcat acatagtttcttagtgtaag catccctgac 600 tgtcttgaag acactttgta ggcctcaatc ctgtaagagccttcctctgc ttttcaaatg 660 ctggcatgaa tgttgtacct cactatgacc agcttagtcttcaagtctga gttactggaa 720 aggagttcca agaagactgg ttatattttt catttattattgcattttaa ttaaaattta 780 atttcaccaa aagaatttag actgaccaat tcagagtctgccgtttaaaa gcataaggaa 840 aaagtaggag aaaaacgtga ggctgtctgt ggatggtcgaggctgcttta gggagcctcg 900 tcaccattct gcacttgcaa accgggccac tagaacccggtgaagggaga aaccaaagcg 960 acctggaaac aataggtcac atgaaggcca gccacctccatcttgttgtg cgggagttca 1020 gttagcagac aagatggctg ccatgcacat gttgtctttcagcttggtga ggtcaaagta 1080 caaccgagtc acagaacaag gaagtataca cagtgagttccaggtcagcc agagtttaca 1140 cagagaaacc acatcttgaa aaaaacaaaa aaataaattaaataaatata atttaaaaat 1200 ttaaaaatag ccgggagtga tggcgcatgt ctttaatcccagctctcttc aggcagagat 1260 gggaggattt ctgagtttga ggccagcctg gtctgcaaagtgagttccag gacagtcagg 1320 gctatacaga gaaaccctgt cttgaaaact aaactaaattaaactaaact aaactaaaaa 1380 aatataaaat aaaaatttta aagaatttta aaaaactacagaaatcaaac ataagcccac 1440 gagatggcaa gtaactgcaa tcatagcaga aatattatacacacacacac acacagactc 1500 tgtcataaaa tccaatgtgc cttcatgatg atcaaatttcgatagtcagt aatactagaa 1560 gaatcatatg tctgaaaata aaagccagaa ccttttctgcttttgttttc ttttgcccca 1620 agatagggtt tctctcagtg tatccctggc atccctgcctggaacttcct ttgtaggttt 1680 ggtagcctca aactcagaga ggtcctctct gcctgcctgcctgcctgcct gcctgcctgc 1740 ctgcctgcct gcctgcctca cttcttctgc cacccacacaaccgagtcga acctaggatc 1800 tttatttctt tctctttctc tcttctttct ttctttctttctttctttct ttctttcttt 1860 ctttctttct ttcttattca attagttttc aatgtaagtgtgtgtttgtg ctctatctgc 1920 tgcctatagg cctgcttgcc aggagagggc aacagaacctaggagaaacc accatgcagc 1980 tcctgagaat aagtgaaaaa acaacaaaaa aaggaaattctaatcacata gaatgtagat 2040 atatgccgag gctgtcagag tgctttttaa ggcttagtgtaagtaatgaa aattgttgtg 2100 tgtcttttat ccaaacacag aagagaggtg gctcggcctgcatgtctgtt gtctgcatgt 2160 agaccaggct ggccttgaac acattaatct gtctgcctctgcttccctaa tgctgcgatt 2220 aaaggcatgt gccaccactg cccggactga tttcttcttttttttttttt tggaaaatac 2280 ctttctttct ttttctctct ctctttcttc cttccttcctttctttctat tctttttttc 2340 tttctttttt cttttttttt ttttttttaa aatttgcctaaggttaaagg tgtgctccac 2400 aattgcctca gctctgctct aattctcttt aaaaaaaaacaaacaaaaaa aaaaccaaaa 2460 cagtatgtat gtatgtatat ttagaagaaa tactaatccattaataactc ttttttccta 2520 aaattcatgt cattcttgtt ccacaaagtg agttccaggacttaccagag aaaccctgtg 2580 ttcaaatttc tgtgttcaag gtcaccctgg cttacaaagtgagttccaag tccgataggg 2640 ctacacagaa aaaccatatc tcagaaaaaa aaaaagttccaaacacacac acacacacac 2700 acacacacac acacacacac acacacacac acacacacagcgcgccgcgg cgatgagggg 2760 aagtcgtgcc taaaataaat atttttctgg ccaaagtgaaagcaaatcac tatgaagagg 2820 tactcctaga aaaaataaat acaaacgggc tttttaatcattccagcact gttttaattt 2880 aactctgaat ttagtcttgg aaaagggggc gggtgtgggtgagtgagggc gagcgagcag 2940 acgggcgggc gggcgggtga gtggccggcg gcggtggcagcgagcaccag aaaacaacaa 3000 accccaagcg gtagagtgtt ttaaaaatga gacctaaatgtggtggaacg gaggtcgccg 3060 ccaccctcct cttccactgc ttagatgctc ccttccccttactgtgctcc cttcccctaa 3120 ctgtgcctaa ctgtgcctgt tccctcaccc cgctgattcgccagcgacgt actttgactt 3180 caagaacgat tttgcctgtt ttcaccgctc cctgtcatactttcgttttt gggtgcccga 3240 gtctagcccg ttcgctatgt tcgggcggga cgatggggaccgtttgtgcc actcgggaga 3300 agtggtgggt gggtacgctg ctccgtcgtg cgtgcgtgagtgccggaacc tgagctcggg 3360 agaccctccg gagagacaga atgagtgagt gaatgtggcggcgcgtgacg gatctgtatt 3420 ggtttgtatg gttgatcgag accattgtcg ggcgacacctagtggtgaca agtttcggga 3480 acgctccagg cctctcaggt tggtgacaca ggagagggaagtgcctgtgg tgaggcgacc 3540 agggtgacag gaggccgggc aagcaggcgg gagcgtctcggagatggtgt cgtgtttaag 3600 gacggtctct aacaaggagg tcgtacaggg agatggccaaagcagaccga gttgctgtac 3660 gcccttttgg gaaaaatgct agggttggtg gcaacgttactaggtcgacc agaaggctta 3720 agtcctaccc ccccccccct tttttttttt tttcctccagaagccctctc ttgtccccgt 3780 caccgggggc accgtacatc tgaggccgag aggacgcgatgggcccggct tccaagccgg 3840 tgtggctcgg ccagctggcg cttcgggtct tttttttttttttttttttt ttttcctcca 3900 gaagccttgt ctgtcgctgt caccgggggc gctgtacttctgaggccgag aggacgcgat 3960 gggccccggc ttccaagccg gtgtggctcg gccagctggagcttcgggtc tttttttttt 4020 tttttttttt tttttttctc cagaagcctt gtctgtcgctgtcaccgggg gcgctgtact 4080 tctgaggccg agaggacgcg atgggtcggc ttccaagccgatgtggcggg gccagctgga 4140 gcttcgggtt tttttttttc ctccagaagc cctctcttgtccccgtcacc gggggcgctg 4200 tacttctgag gccgagagga cgtgatgggc ccgggttccaggcggatgtc gcccggtcag 4260 ctggagcttt ggatcttttt tttttttttt cctccagaagccctctcttg tccccgtcac 4320 cgggggcacc ttacatctga gggcgagagg acgtgatgggtccggcttcc aagccgatgt 4380 ggcggggcca gctggagctt cgggtttttt ttttttcctccagaagccct ctcttgtccc 4440 cgtcaccggg ggcgctgtac ttctgaggcc gagaggacgtgatgggcccg ggttccaggc 4500 ggatgtcgcc cggtcagctg gagctttgga tcatttttttttttccctcc agaagccctc 4560 tcttgtcccc gtcaccgggg gcaccgtaca tctgaggccgagaggacacg atgggcctgt 4620 cttccaagcc gatgtggccc ggccagctgg agcttcgggtcttttttttt ttttttcctc 4680 cagaagcctt gtctgtcgct gtcacccggg gcgctgtacttctgaggccg agaggacgcg 4740 atgggcccgg cttccaagcc ggtgtggctc ggccagctggagcttcgggt cttttttttt 4800 tttttttttt ttcctccaga aaccttgtct gtcgctgtcacccggggcgc ttgtacttct 4860 gatgccgaga ggacgcgatg ggcccgtctt ccaggccgatgtggcccggt cagctggagc 4920 tttggatctt tttttttttt ttttcctcca gaagccctctcttgtccccg tcaccggggg 4980 caccttacat ctgaggccta gaggacacga tgggcccgggttccaggccg atgtggcccg 5040 gtcagctgga gctttggatc tttttttttt ttttcttccagaagccctct tgtccccgtc 5100 accggtggca ctgtacatct gaggcggaga ggacattatgggcccggctt ccaatccgat 5160 gtggcccggt cagctggagc tttggatctt attttttttttaattttttc ttccagaagc 5220 cctcttgtcc ctgtcaccgg tggcacggta catctgaggccgagaggaca ttatgggccc 5280 ggcttccagg ccgatgtggc ccggtcagct ggagctttggatcttttttt ttttttttct 5340 tttttcctcc agaagccctc tctgtccctg tcaccgggggccctgtacgt ctgaggccga 5400 gggaaagcta tgggcgcggt tttctttcat tgacctgtcggtcttatcag ttctccgggt 5460 tgtcagggtc gaccagttgt tcctttgagg tccggttcttttcgttatgg ggtcattttt 5520 gggccacctc cccaggtatg acttccaggc gtcgttgctcgcctgtcact ttcctccctg 5580 tctcttttat gcttgtgatc ttttctatct gttcctattggacctggaga taggtactga 5640 cacgctgtcc tttccctatt aacactaaag gacactataaagagaccctt tcgatttaag 5700 gctgttttgc ttgtccagcc tattcttttt actggcttgggtctgtcgcg gtgcctgaag 5760 ctgtccccga gccacgcttc ctgctttccc gggcttgctgcttgcgtgtg cttgctgtgg 5820 gcagcttgtg acaactgggc gctgtgactt tgctgcgtgtcagacgtttt tcccgatttc 5880 cccgaggtgt cgttgtcaca cctgtcccgg ttggaatggtggagccagct gtggttgagg 5940 gccaccttat ttcggctcac tttttttttt tttttttctcttggagtccc gaacctccgc 6000 tcttttctct tcccggtctt tcttccacat gcctcccgagtgcatttctt tttgtttttt 6060 ttcttttttt tttttttttt ttggggaggt ggagagtcccgagtacttca ctcctgtctg 6120 tggtgtccaa gtgttcatgc cacgtgcctc ccgagtgcacttttttttgt ggcagtcgct 6180 cgttgtgttc tcttgttctg tgtctgcccg tatcagtaactgtcttgccc cgcgtgtaag 6240 acattcctat ctcgcttgtt tctcccgatt gcgcgtcgttgctcactctt agatcgatgt 6300 ggtgctccgg agttctcttc gggccagggc caagccgcgccaggcgaggg acggacattc 6360 atggcgaatg gcggccgctc ttctcgttct gccagcgggccctcgtctct ccaccccatc 6420 cgtctgccgg tggtgtgtgg aaggcagggg tgcggctctccggcccgacg ctgccccgcg 6480 cgcacttttc tcagtggttc gcgtggtcct tgtggatgtgtgaggcgccc ggttgtgccc 6540 tcacgtgttt cactttggtc gtgtctcgct tgaccatgttcccagagtcg gtggatgtgg 6600 ccggtggcgt tgcataccct tcccgtctgg tgtgtgcacgcgctgtttct tgtaagcgtc 6660 gaggtgctcc tggagcgttc caggtttgtc tcctaggtgcctgcttctga gctggtggtg 6720 gcgctcccca ttccctggtg tgcctccggt gctccgtctggctgtgtgcc ttcccgtttg 6780 tgtctgagaa gcccgtgaga ggggggtcga ggagagaaggaggggcaaga ccccccttct 6840 tcgtcgggtg aggcgcccac cccgcgacta gtacgcctgtgcgtagggct ggtgctgagc 6900 ggtcgcggct ggggttggaa agtttctcga gagactcattgctttcccgt ggggagcttt 6960 gagaggcctg gctttcgggg gggaccggtt gcagggtctcccctgtccgc ggatgctcag 7020 aatgcccttg gaagagaacc ttcctgttgc cgcagacccccccgcgcggt cgcccgcgtg 7080 ttggtcttct ggtttccctg tgtgctcgtc gcatgcatcctctctcggtg gccggggctc 7140 gtcggggttt tgggtccgtc ccgccctcag tgagaaagtttccttctcta gctatcttcc 7200 ggaaagggtg cgggcttctt acggtctcga ggggtctctcccgaatggtc ccctggaggg 7260 ctcgccccct gaccgcctcc cgcgcgcgca gcgtttgctctctcgtctac cgcggcccgc 7320 ggcctccccg ctccgagttc ggggagggat cacgcggggcagagcctgtc tgtcgtcctg 7380 ccgttgctgc ggagcatgtg gctcggcttg tgtggttggtggctggggag agggctccgt 7440 gcacaccccc gcgtgcgcgt actttcctcc cctcctgagggccgccgtgc ggacggggtg 7500 tgggtaggcg acggtgggct cccgggtccc cacccgtcttcccgtgcctc acccgtgcct 7560 tccgtcgcgt gcgtccctct cgctcgcgtc cacgactttggccgctcccg cgacggcggc 7620 ctgcgccgcg cgtggtgcgt gctgtgtgct tctcgggctgtgtggttgtg tcgcctcgcc 7680 ccccccttcc cgcggcagcg ttcccacggc tggcgaaatcgcgggagtcc tccttcccct 7740 cctcggggtc gagagggtcc gtgtctggcg ttgattgatctcgctctcgg ggacgggacc 7800 gttctgtggg agaacggctg ttggccgcgt ccggcgcgacgtcggacgtg gggacccact 7860 gccgctcggg ggtcttcgtc ggtaggcatc ggtgtgtcggcatcggtctc tctctcgtgt 7920 cggtgtcgcc tcctcgggct cccggggggc cgtcgtgtttcgggtcggct cggcgctgca 7980 ggtgtggtgg gactgctcag gggagtggtg cagtgtgattcccgccggtt ttgcctcgcg 8040 tgccctgacc ggtccgacgc ccgagcggtc tctcggtcccttgtgaggac ccccttccgg 8100 gaggggcccg tttcggccgc ccttgccgtc gtcgccggccctcgttctgc tgtgtcgttc 8160 ccccctcccc gctcgccgca gccggtcttt tttcctctctccccccctct cctctgactg 8220 acccgtggcc gtgctgtcgg accccccgca tgggggcggccgggcacgta cgcgtccggg 8280 cggtcaccgg ggtcttgggg gggggccgag gggtaagaaagtcggctcgg cgggcgggag 8340 gagctgtggt ttggagggcg tcccggcccc gcggccgtggcggtgtcttg cgcggtcttg 8400 gagagggctg cgtgcgaggg gaaaaggttg ccccgcgagggcaaagggaa agaggctagc 8460 agtggtcatt gtcccgacgg tgtggtggtc tgttggccgaggtgcgtctg gggggctcgt 8520 ccggccctgt cgtccgtcgg gaaggcgcgt gttggggcctgccggagtgc cgaggtgggt 8580 accctggcgg tgggattaac cccgcgcgcg tgtcccggtgtggcggtggg ggctccggtc 8640 gatgtctacc tccctctccc cgaggtctca ggccttctccgcgcgggctc tcggccctcc 8700 cctcgttcct ccctctcgcg gggttcaagt cgctcgtcgacctcccctcc tccgtccttc 8760 catctctcgc gcaatggcgc cgcccgagtt cacggtgggttcgtcctccg cctccgcttc 8820 tcgccggggg ctggccgctg tccggtctct cctgcccgacccccgttggc gtggtcttct 8880 ctcgccggct tcgcggactc ctggcttcgc ccggagggtcagggggcttc ccggttcccc 8940 gacgttgcgc ctcgctgctg tgtgcttggg gggggcccgctgcggcctcc gcccgcccgt 9000 gagcccctgc cgcacccgcc ggtgtgcggt ttcgcgccgcggtcagttgg gccctggcgt 9060 tgtgtcgcgt cgggagcgtg tccgcctcgc ggcggctagacgcgggtgtc gccgggctcc 9120 gacgggtggc ctatccaggg ctcgcccccg ccgacccccgcctgcccgtc ccggtggtgg 9180 tcgttggtgt ggggagtgaa tggtgctacc ggtcattccctcccgcgtgg tttgactgtc 9240 tcgccggtgt cgcgcttctc tttccgccaa cccccacgccaacccaccac cctgctctcc 9300 cggcccggtg cggtcgacgt tccggctctc ccgatgccgaggggttcggg atttgtgccg 9360 gggacggagg ggagagcggg taagagaggt gtcggagagctgtcccgggg cgacgctcgg 9420 gttggctttg ccgcgtgcgt gtgctcgcgg acgggttttgtcggaccccg acggggtcgg 9480 tccggccgca tgcactctcc cgttccgcgc gagcgcccgcccggctcacc cccggtttgt 9540 cctcccgcga ggctctccgc cgccgccgcc tcctcctcctctctcgcgct ctctgtcccg 9600 cctggtcctg tcccaccccc gacgctccgc tcgcgcttccttacctggtt gatcctgcca 9660 ggtagcatat gcttgtctca aagattaagc catgcatgtctaagtacgca cggccggtac 9720 agtgaaactg cgaatggctc attaaatcag ttatggttcctttggtcgct cgctcctctc 9780 ctacttggat aactgtggta attctagagc taatacatgccgacgggcgc tgacccccct 9840 tcccgggggg ggatgcgtgc atttatcaga tcaaaaccaacccggtgagc tccctcccgg 9900 ctccggccgg gggtcgggcg ccggcggctt ggtgactctagataacctcg ggccgatcgc 9960 acgccccccg tggcggcgac gacccattcg aacgtctgccctatcaactt tcgatggtag 10020 tcgccgtgcc taccatggtg accacgggtg acggggaatcagggttcgat tccggagagg 10080 gagcctgaga aacggctacc acatccaagg aaggcagcaggcgcgcaaat tacccactcc 10140 cgacccgggg aggtagtgac gaaaaataac aatacaggactctttcgagg ccctgtaatt 10200 ggaatgagtc cactttaaat cctttaacga ggatccattggagggcaagt ctggtgccag 10260 cagccgcggt aattccagct ccaatagcgt atattaaagttgctgcagtt aaaaagctcg 10320 tagttggatc ttgggagcgg gcgggcggtc cgccgcgaggcgagtcaccg cccgtccccg 10380 ccccttgcct ctcggcgccc cctcgatgct cttagctgagtgtcccgcgg ggcccgaagc 10440 gtttactttg aaaaaattag agtgttcaaa gcaggcccgagccgcctgga taccgcagct 10500 aggaataatg gaataggacc gcggttctat tttgttggttttcggaactg aggccatgat 10560 taagagggac ggccgggggc attcgtattg cgccgctagaggtgaaattc ttggaccggc 10620 gcaagacgga ccagagcgaa agcatttgcc aagaatgttttcattaatca agaacgaaag 10680 tcggaggttc gaagacgatc agataccgtc gtagttccgaccataaacga tgccgactgg 10740 cgatgcggcg gcgttattcc catgacccgc cgggcagcttccgggaaacc aaagtctttg 10800 ggttccgggg ggagtatggt tgcaaagctg aaacttaaaggaattgacgg aagggcacca 10860 ccaggagtgg gcctgcggct taatttgact caacacgggaaacctcaccc ggcccggaca 10920 cggacaggat tgacagattg atagctcttt ctcgattccgtgggtggtgg tgcatggccg 10980 ttcttagttg gtggagcgat ttgtctggtt aattccgataacgaacgaga ctctggcatg 11040 ctaactagtt acgcgacccc cgagcggtcg gcgtcccccaacttcttaga gggacaagtg 11100 gcgttcagcc acccgagatt gagcaataac aggtctgtgatgcccttaga tgtccggggc 11160 tgcacgcgcg ctacactgac tggctcagcg tgtgcctaccctgcgccggc aggcgcgggt 11220 aacccgttga accccattcg tgatggggat cggggattgcaattattccc catgaacgag 11280 gaattcccag taagtgcggg tcataagctt gcgttgattaagtccctgcc ctttgtacac 11340 accgcccgtc gctactaccg attggatggt ttagtgaggccctcggatcg gccccgccgg 11400 ggtcggccca cggccctggc ggagcgctga gaagacggtcgaacttgact atctagagga 11460 agtaaaagtc gtaacaaggt ttccgtaggt gaacctgcggaaggatcatt aaacgggaga 11520 ctgtggagga gcggcggcgt ggcccgctct ccccgtcttgtgtgtgtcct cgccgggagg 11580 cgcgtgcgtc ccgggtcccg tcgcccgcgt gtggagcgaggtgtctggag tgaggtgaga 11640 gaaggggtgg gtggggtcgg tctgggtccg tctgggaccgcctccgattt cccctccccc 11700 tcccctctcc ctcgtccggc tctgacctcg ccaccctaccgcggcggcgg ctgctcgcgg 11760 gcgtcttgcc tctttcccgt ccggctcttc cgtgtctacgaggggcggta cgtcgttacg 11820 ggtttttgac ccgtcccggg ggcgttcggt cgtcggggcgcgcgctttgc tctcccggca 11880 cccatccccg ccgcggctct ggcttttcta cgttggctggggcggttgtc gcgtgtgggg 11940 ggatgtgagt gtcgcgtgtg ggctcgcccg tcccgatgccacgcttttct ggcctcgcgt 12000 gtcctccccg ctcctgtccc gggtacctag ctgtcgcgttccggcgcgga ggtttaagga 12060 ccccgggggg gtcgccctgc cgcccccagg gtcggggggcggtggggccc gtagggaagt 12120 cggtcgttcg ggcggctctc cctcagactc catgaccctcctccccccgc tgccgccgtt 12180 cccgaggcgg cggtcgtgtg ggggggtgga tgtctggagccccctcgggc gccgtggggg 12240 cccgacccgc gccgccggct tgcccgattt ccgcgggtcggtcctgtcgg tgccggtcgt 12300 gggttcccgt gtcgttcccg tgtttttccg ctcccgaccctttttttttc ctccccccca 12360 cacgtgtctc gtttcgttcc tgctggccgg cctgaggctacccctcggtc catctgttct 12420 cctctctctc cggggagagg agggcggtgg tcgttgggggactgtgccgt cgtcagcacc 12480 cgtgagttcg ctcacacccg aaataccgat acgactcttagcggtggatc actcggctcg 12540 tgcgtcgatg aagaacgcag ctagctgcga gaattaatgtgaattgcagg acacattgat 12600 catcgacact tcgaacgcac ttgcggcccc gggttcctcccggggctacg cctgtctgag 12660 cgtcggttga cgatcaatcg cgtcacccgc tgcggtgggtgctgcgcggc tgggagtttg 12720 ctcgcagggc caacccccca acccgggtcg ggccctccgtctcccgaagt tcagacgtgt 12780 gggcggttgt cggtgtggcg cgcgcgcccg cgtcgcggagcctggtctcc cccgcgcatc 12840 cgcgctcgcg gcttcttccc gctccgccgt tcccgccctcgcccgtgcac cccggtcctg 12900 gcctcgcgtc ggcgcctccc ggaccgctgc ctcaccagtctttctcggtc ccgtgccccg 12960 tgggaaccca ccgcgccccc gtggcgcccg ggggtgggcgcgtccgcatc tgctctggtc 13020 gaggttggcg gttgagggtg tgcgtgcgcc gaggtggtggtcggtcccct gcggccgcgg 13080 ggttgtcggg gtggcggtcg acgagggccg gtcggtcgcctgcggtggtt gtctgtgtgt 13140 gtttgggtct tgcgctgggg gaggcggggt cgaccgctcgcggggttggc gcggtcgccc 13200 ggcgccgcgc accctccggc ttgtgtggag ggagagcgagggcgagaacg gagagaggtg 13260 gtatccccgg tggcgttgcg agggagggtt tggcgtcccgcgtccgtccg tccctccctc 13320 cctcggtggg cgccttcgcg ccgcacgcgg ccgctaggggcggtcggggc ccgtggcccc 13380 cgtggctctt cttcgtctcc gcttctcctt cacccgggcggtacccgctc cggcgccggc 13440 ccgcgggacg ccgcggcgtc cgtgcgccga tgcgagtcacccccgggtgt tgcgagttcg 13500 gggagggaga gggcctcgct gacccgttgc gtcccggcttccctgggggg gacccggcgt 13560 ctgtgggctg tgcgtcccgg gggttgcgtg tgagtaagatcctccacccc cgccgccctc 13620 ccctcccgcc ggcctctcgg ggaccccctg agacggttcgccggctcgtc ctcccgtgcc 13680 gccgggtgcc gtctctttcc cgcccgcctc ctcgctctcttcttcccgcg gctgggcgcg 13740 tgtcccccct ttctgaccgc gacctcagat cagacgtggcgacccgctga atttaagcat 13800 attagtcagc ggaggaaaag aaactaacca ggattccctcagtaacggcg agtgaacagg 13860 gaagagccca gcgccgaatc cccgccgcgc gtcgcggcgtgggaaatgtg gcgtacggaa 13920 gacccactcc ccggcgccgc tcgtgggggg cccaagtccttctgatcgag gcccagcccg 13980 tggacggtgt gaggccggta gcggccccgg cgcgccgggctcgggtcttc ccggagtcgg 14040 gttgcttggg aatgcagccc aaagcgggtg gtaaactccatctaaggcta aataccggca 14100 cgagaccgat agtcaacaag taccgtaagg gaaagttgaaaagaactttg aagagagagt 14160 tcaagagggc gtgaaaccgt taagaggtaa acgggtggggtccgcgcagt ccgcccggag 14220 gattcaaccc ggcggcgcgc gtccggccgt gcccggtggtcccggcggat ctttcccgct 14280 ccccgttcct cccgacccct ccacccgcgc gtcgttcccctcttcctccc cgcgtccggc 14340 gcctccggcg gcgggcgcgg ggggtggtgt ggtggtggcgcgcgggcggg gccgggggtg 14400 gggtcggcgg gggaccgccc ccggccggcg accggccgccgccgggcgca cttccaccgt 14460 ggcggtgcgc cgcgaccggc tccgggacgg ccgggaaggcccggtgggga aggtggctcg 14520 gggggggcgg cgcgtctcag ggcgcgccga accacctcaccccgagtgtt acagccctcc 14580 ggccgcgctt tcgccgaatc ccggggccga ggaagccagatacccgtcgc cgcgctctcc 14640 ctctcccccc gtccgcctcc cgggcgggcg tgggggtgggggccgggccg cccctcccac 14700 ggcgcgaccg ctctcccacc cccctccgtc gcctctctcggggcccggtg gggggcgggg 14760 cggactgtcc ccagtgcgcc ccgggcgtcg tcgcgccgtcgggtcccggg gggaccgtcg 14820 gtcacgcgtc tcccgacgaa gccgagcgca cggggtcggcggcgatgtcg gctacccacc 14880 cgacccgtct tgaaacacgg accaaggagt ctaacgcgtgcgcgagtcag gggctcgtcc 14940 gaaagccgcc gtggcgcaat gaaggtgaag ggccccgcccgggggcccga ggtgggatcc 15000 cgaggcctct ccagtccgcc gagggcgcac caccggcccgtctcgcccgc cgcgccgggg 15060 aggtggagca cgagcgtacg cgttaggacc cgaaagatggtgaactatgc ttgggcaggg 15120 cgaagccaga ggaaactctg gtggaggtcc gtagcggtcctgacgtgcaa atcggtcgtc 15180 cgacctgggt ataggggcga aagactaatc gaaccatctagtagctggtt ccctccgaag 15240 tttccctcag gatagctggc gctctcgctc ccgacgtacgcagttttatc cggtaaagcg 15300 aatgattaga ggtcttgggg ccgaaacgat ctcaacctattctcaaactt taaatgggta 15360 agaagcccgg ctcgctggcg tggagccggg cgtggaatgcgagtgcctag tgggccactt 15420 ttggtaagca gaactggcgc tgcgggatga accgaacgccgggttaaggc gcccgatgcc 15480 gacgctcatc agaccccaga aaaggtgttg gttgatatagacagcaggac ggtggccatg 15540 gaagtcggaa tccgctaagg agtgtgtaac aactcacctgccgaatcaac tagccctgaa 15600 aatggatggc gctggagcgt cgggcccata cccggccgtcgccgcagtcg gaacggaacg 15660 ggacgggagc ggccgcgggt gcgcgtctct cggggtcgggggtgcgtggc gggggcccgt 15720 cccccgcctc ccctccgcgc gccgggttcg cccccgcggcgtcgggcccc gcggagccta 15780 cgccgcgacg agtaggaggg ccgctgcggt gagccttgaagcctagggcg cgggcccggg 15840 tggagccgcc gcaggtgcag atcttggtgg tagtagcaaatattcaaacg agaactttga 15900 aggccgaagt ggagaagggt tccatgtgaa cagcagttgaacatgggtca gtcggtcctg 15960 agagatgggc gagtgccgtt ccgaagggac gggcgatggcctccgttgcc ctcggccgat 16020 cgaaagggag tcgggttcag atccccgaat ccggagtggcggagatgggc gccgcgaggc 16080 cagtgcggta acgcgaccga tcccggagaa gccggcgggaggcctcgggg agagttctct 16140 tttctttgtg aagggcaggg cgccctggaa tgggttcgccccgagagagg ggcccgtgcc 16200 ttggaaagcg tcgcggttcc ggcggcgtcc ggtgagctctcgctggccct tgaaaatccg 16260 ggggagaggg tgtaaatctc gcgccgggcc gtacccatatccgcagcagg tctccaaggt 16320 gaacagcctc tggcatgttg gaacaatgta ggtaagggaagtcggcaagc cggatccgta 16380 acttcgggat aaggattggc tctaagggct gggtcggtcgggctggggcg cgaagcgggg 16440 ctgggcgcgc gccgcggctg gacgaggcgc cgccgccctctcccacgtcc ggggagaccc 16500 cccgtccttt ccgcccgggc ccgccctccc ctcttccccgcggggccccg tcgtcccccg 16560 cgtcgtcgcc acctctcttc ccccctcctt cttcccgtcggggggcgggt cgggggtcgg 16620 cgcgcggcgc gggctccggg gcggcgggtc caaccccgcgggggttccgg agcgggagga 16680 accagcggtc cccggtgggg cggggggccc ggacactcggggggccggcg gcggcggcga 16740 ctctggacgc gagccgggcc cttcccgtgg atcgcctcagctgcggcggg cgtcgcggcc 16800 gctcccgggg agcccggcgg gtgccggcgc gggtcccctccccgcggggc ctcgctccac 16860 ccccccatcg cctctcccga ggtgcgtggc gggggcgggcgggcgtgtcc cgcgcgtgtg 16920 gggggaacct ccgcgtcggt gttcccccgc cgggtccgccccccgggccg cggttttccg 16980 cgcggcgccc ccgcctcggc cggcgcctag cagccgacttagaactggtg cggaccaggg 17040 gaatccgact gtttaattaa aacaaagcat cgcgaaggcccgcggcgggt gttgacgcga 17100 tgtgatttct gcccagtgct ctgaatgtca aagtgaagaaattcaatgaa gcgcgggtaa 17160 acggcgggag taactatgac tctcttaagg tagccaaatgcctcgtcatc taattagtga 17220 cgcgcatgaa tggatgaacg agattcccac tgtccctacctactatccag cgaaaccaca 17280 gccaagggaa cgggcttggc ggaatcagcg gggaaagaagaccctgttga gcttgactct 17340 agtctggcac ggtgaagaga catgagaggt gtagaataagtgggaggccc ccggcgcccg 17400 gccccgtcct cgcgtcgggg tcggggcacg ccggcctcgcgggccgccgg tgaaatacca 17460 ctactctcat cgttttttca ctgacccggt gaggcgggggggcgagcccc gaggggctct 17520 cgcttctggc gccaagcgtc cgtcccgcgc gtgcgggcgggcgcgacccg ctccggggac 17580 agtgccaggt ggggagtttg actggggcgg tacacctgtcaaacggtaac gcaggtgtcc 17640 taaggcgagc tcagggagga cagaaacctc ccgtggagcagaagggcaaa agctcgcttg 17700 atcttgattt tcagtacgaa tacagaccgt gaaagcggggcctcacgatc cttctgacct 17760 tttgggtttt aagcaggagg tgtcagaaaa gttaccacagggataactgg cttgtggcgg 17820 ccaagcgttc atagcgacgt cgctttttga tccttcgatgtcggctcttc ctatcattgt 17880 gaagcagaat tcaccaagcg ttggattgtt cacccactaatagggaacgt gagctgggtt 17940 tagaccgtcg tgagacaggt tagttttacc ctactgatgatgtgttgttg ccatggtaat 18000 cctgctcagt acgagaggaa ccgcaggttc agacatttggtgtatgtgct tggctgagga 18060 gccaatgggg cgaagctacc atctgtggga ttatgactgaacgcctctaa gtcagaatcc 18120 gcccaagcgg aacgatacgg cagcgccgaa ggagcctcggttggccccgg atagccgggt 18180 ccccgtccgt cccgctcggc ggggtccccg cgtcgccccgcggcggcgcg gggtctcccc 18240 ccgccgggcg tcgggaccgg ggtccggtgc ggagagccgttcgtcttggg aaacggggtg 18300 cggccggaaa gggggccgcc ctctcgcccg tcacgttgaacgcacgttcg tgtggaacct 18360 ggcgctaaac cattcgtaga cgacctgctt ctgggtcggggtttcgtacg tagcagagca 18420 gctccctcgc tgcgatctat tgaaagtcag ccctcgacacaagggtttgt ctctgcgggc 18480 tttcccgtcg cacgcccgct cgctcgcacg cgaccgtgtcgccgcccggg cgtcacgggg 18540 gcggtcgcct cggcccccgc gcggttgccc gaacgaccgtgtggtggttg ggggggggat 18600 cgtcttctcc tccgtctccc gaggacggtt cgtttctctttccccttccg tcgctctcct 18660 tgggtgtggg agcctcgtgc cgtcgcgacc gcggcctgccgtcgcctgcc gccgcagccc 18720 cttgccctcc ggccttggcc aagccggagg gcggaggagggggatcggcg gcggcggcga 18780 ccgcggcgcg gtgacgcacg gtgggatccc catcctcggcgcgtccgtcg gggacggccg 18840 gttggagggg cgggaggggt ttttcccgtg aacgccgcgttcggcgccag gcctctggcg 18900 gccggggggg cgctctctcc gcccgagcat ccccactcccgcccctcctc ttcgcgcgcc 18960 gcggcggcga cgtgcgtacg aggggaggat gtcgcggtgtggaggcggag agggtccggc 19020 gcggcgcctc ttccattttt tcccccccaa cttcggaggtcgaccagtac tccgggcgac 19080 actttgtttt ttttttttcc cccgatgctg gaggtcgaccagatgtccga aagtgtcccc 19140 cccccccccc ccccccggcg cggagcggcg gggccactctggactctttt tttttttttt 19200 tttttttttt ttaaattcct ggaaccttta ggtcgaccagttgtccgtct tttactcctt 19260 catataggtc gaccagtact ccgggtggta ctttgtctttttctgaaaat cccagaggtc 19320 gaccagatat ccgaaagtcc tctctttccc tttactcttccccacagcga ttctcttttt 19380 tttttttttt tttggtgtgc ctctttttga cttatatacatgtaaatagt gtgtacgttt 19440 atatacttat aggaggaggt cgaccagtac tccgggcgacactttgtttt tttttttttt 19500 tccaccgatg atggaggtcg accagatgtc cgaaagtgtcccgtcccccc cctccccccc 19560 ccgcgacgcg gcgggctcac tctggactct tttttttttttttttttttt tttaaatttc 19620 tggaacctta aggtcgacca gttgtccgtc tttcactcattcatataggt cgaccggtgg 19680 tactttgtct ttttctgaaa atcgcagagg tcgaccagatgtcagaaagt ctggtggtcg 19740 ataaattatc tgatctagat ttgtttttct gtttttcagttttgtgttgt tttgtgttgt 19800 tttgtgttgt tttgttttgt tttgttttgt tttgttttgttttgttttgt tttgttttgt 19860 tttgtgttgt gttgtgttgt gttgtgttgg gttgggttgggttgggttgg gttgggttgg 19920 gttgggttgg gttgggttgt gttgtttggt tttgtgttgtttggtgttgt tggttttgtt 19980 ttgtttgctg ttgttttgtg ttttgcgggt cgaacagttgtccctaaccg agtttttttg 20040 tacacaaaca tgcacttttt ttaaaataaa tttttaaaataaatgcgaaa atcgaccaat 20100 tatccctttc cttctctctc ttttttaaaa attttctttgtgtgtgtgtg tgtgtgtgtg 20160 tgtgtgtgtg tgcgtgtgtg tgtgtgtgtg cgtgcagcgtgcgcgcgctc gttttataaa 20220 tacttataat aataggtcgc cgggtggtgg tagcttcccggactccagag gcagaggcag 20280 gcagacttct gagttcgagg ccagcctggt ctacagaggaaccctgtctc gaaaaatgaa 20340 aataaataca tacatacata catacataca tacatacatacatacataca tacatatgag 20400 gttgaccagt tgtcaatcct ttagaatttt gtttttaattaatgtgatag agagatagat 20460 aatagataga tggatagagt gatacaaata taggtttttttttcagtaaa tatgaggttg 20520 attaaccact tttccctttt taggtttttt tttttttcccctgtccatgt ggttgctggg 20580 atttgaactc aggaccctgg caggtcaact ggaaaacgtgttttctatat atataaatag 20640 tggtctgtct gctgtttgtt tgtttgcttg cttgcttgcttgcttgcttg cttgcttgct 20700 tgcttttttt tttcttctga gacagtattt ctctgtgtaacctggtgccc tgaaactcac 20760 tctgtagacc agcctggcct caatcgaact cagaaatcctcctgcctctt gtctacctcc 20820 caattttgga gtaaaggtgt gctacaccac tgcctggcattattatcatt atcattatta 20880 attttattat tagacagaac gaaatcaact agttggtcctgtttcgttaa ttcatttgaa 20940 attagttgga ccaattagtt ggctggtttg ggaggtttcttttgtttccg atttgggtgt 21000 ttgtggggct ggggatcagg tatctcaacg gaatgcatgaaggttaaggt gagatggctc 21060 gatttttgta aagattactt ttcttagtct gaggaaaaaataaaataata ttgggctacg 21120 tttcattgct tcatttctat ttctctttct ttctttctttctttcagata aggaggtcgg 21180 ccagttcctc ctgccttctg gaagatgtag gcattgcattgggaaaagca ttgtttgaga 21240 gatgtgctag tgaaccagag agtttggatg tcaagccgtataatgtttat tacaatatag 21300 aaaagttcta acaaagtgat ctttaacttt tttttttttttttctccttc tacttctact 21360 tgttctcact ctgccaccaa cgcgctttgt acattgaatgtgagctttgt tttgcttaac 21420 agacatatat tttttctttt ggttttgctt gacatggtttccctttctat ccgtgcaggg 21480 ttcccagacg gccttttgag aataaaatgg gaggccagaaccaaagtctt ttgaataaag 21540 caccacaact ctaacctgtt tggctgtttt ccttcccaaggcacagatct ttcccagcat 21600 ggaaaagcat gtagcagttg taggacacac tagacgagagcaccagatct cattgtgggt 21660 ggttgtgaac cacccaccat gtggttgcct gggatttgaactcaggatct tcagaagacg 21720 agtcagggct ctaaaccgat gagccatctc tccagccctcctacattcct tcttaaggca 21780 tgaatgatcc cagcatggga agacagtctg ccctctttgtggtatatcac catatactca 21840 ataaaataat gaaatgaatg aagtctccac gtatttatttcttcgagcta tctaaattct 21900 ctcacagcac ctccccctcc cccacactgc ctttctccctatgtttgggt ggggctgggg 21960 gaggggtggg gtgggggcag ggatctgcat gtcttcttgcaggtctgtga actatttgcg 22020 atggcctggt tctctgaact gttgagcctt gtctatccagaggctgactg gctagttttc 22080 tacctgaagt ccctgagtga tgatttccct gtgaattc22118 6 175 DNA Mus Musculus 6 ctcccgcgcg gcccccgtgt tcgccgttcccgtggcgcgg acaatgcggt tgtgcgtcca 60 cgtgtgcgtg tccgtgcagt gccgttgtggagtgcctcgc tctcctcctc ctccccggca 120 gcgttcccac ggttggggac caccggtgacctcgccctct tcgggcctgg atccg 175 7 755 DNA Mus musculus 7 ggtctggtgggaattgttga cctcgctctc gggtgcggcc tttggggaac ggcggggtcg 60 gtcgtgcccggcgccggacg tgtgtcgggg cccacttccc gctcgagggt ggcggtggcg 120 gcggcgttggtagtctcccg tgttgcgtct tcccgggctc ttgggggggg tgccgtcgtt 180 ttcggggccggcgttgcttg gcttacgcag gcttggtttg ggactgcctc aggagtcgtg 240 ggcggtgtgattcccgccgg ttttgcctcg cgtctgcctg ctttgcctcg ggtttgcttg 300 gttcgtgtctcgggagcggt ggtttttttt tttttcgggt cccggggaga ggggtttttc 360 cgggggacgttcccgtcgcc ccctgccgcc ggtgggtttt cgtttcgggc tgtgttcgtt 420 tccccttccccgtttcgccg tcggttctcc ccggtcggtc ggccctctcc ccggtcggtc 480 gcccggccgtgctgccggac ccccccttct gggggggatg cccgggcacg cacgcgtccg 540 ggcggccactgtggtccggg agctgctcgg caggcgggtg agccagttgg aggggcgtca 600 tgcccccgcgggctcccgtg gccgacgcgg cgtgttcttt gggggggcct gtgcgtgcgg 660 gaaggctgcgcacgttgtcg gtccttgcga gggaaagagg cttttttttt ttagggggtc 720 gtccttcgtcgtcccgtcgg cggtggatcc ggcct 755 8 463 DNA Mus musculus 8 ggccgaggtgcgtctgcggg ttggggctcg tccggccccg tcgtcctccg ggaaggcgtt 60 tagcgggtaccgtcgccgcg ccgaggtggg cgcacgtcgg tgagataacc ccgagcgtgt 120 ttctggttgttggcggcggg ggctccggtc gatgtcttcc cctccccctc tccccgaggc 180 caggtcagcctccgcctgtg ggcttcgtcg gccgtctccc cccccctcac gtccctcgcg 240 agcgagcccgtccgttcgac cttccttccg ccttcccccc atctttccgc gctccgttgg 300 ccccggggttttcacggcgc cccccacgct cctccgcctc tccgcccgtg gtttggacgc 360 ctggttccggtctccccgcc aaaccccggt tgggttggtc tccggccccg gcttgctctt 420 cgggtctcccaacccccggc cggaagggtt cgggggttcc ggg 463 9 378 DNA Mus musculus 9ggattcttca ggattgaaac ccaaaccggt tcagtttcct ttccggctcc ggccgggggg 60ggcggccccg ggcggtttgg tgagttagat aacctcgggc cgatcgcacg ccccccgtgg 120cggcgacgac ccattcgaac gtctgcccta tcaactttcg atggtagtcg atgtgcctac 180catggtgacc acgggtgacg gggaatcagg gttcgattcc ggagagggag cctgagaaac 240ggctaccaca tccaaggaag gcagcaggcg cgcaaattac ccactcccga cccggggagg 300tagtgacgaa aaataacaat acaggactct ttcgaggccc tgtaattgga atgagtccac 360tttaaatcct ttaagcag 378 10 378 DNA Mus musculus 10 gatccattgg agggcaagtctggtgccagc agccgcggta attccagctc caatagcgta 60 tattaaagtt gctgcagttaaaaagctcgt agttggatct tgggagcggg cgggcggtcc 120 gccgcgaggc gagtcaccgcccgtccccgc cccttgcctc tcggcgcccc ctcgatgctc 180 ttagctgagt tgtcccgcggggcccgaagc gtttactttg aaaaaattag agttgtttca 240 aagcaggccc gagccgcctggataccgcca gctaggaaat aatggaatag gaccgcggtt 300 cctattttgt ttggttttcggaactgagcc catgattaag ggaaacggcc gggggcattc 360 ccttattgcg ccccccta 37811 719 DNA Mus musculus 11 ggatctttcc cgctccccgt tcctcccggc ccctccacccgcgcgtctcc ccccttcttt 60 tcccctctcc ggaggggggg gaggtggggg cgcgtgggcggggtcggggg tggggtcggc 120 gggggaccgc ccccggccgg caaaaggccg ccgccgggcgcacttcaacc gtagcggtgc 180 gccgcgaccg gctacgagac ggctgggaag gcccgacggggaatgtggct cggggggggc 240 ggcgcgtctc agggcgcgcc gaaccacctc accccgagtgttacagccct ccggccgcgc 300 tttcgcggaa tcccggggcc gaggggaagc ccgatacccgtcgccgcgct tttcccctcc 360 ccccgtccgc ctcccgggcg ggcgtggggg tgggggccgggccgcccctc ccacgcccgt 420 ggtttctctc tctcccggtc tcggccggtt tgggggggggagcccggttg ggggcggggc 480 ggactgtcct cagtgcgccc cgggcgtcgt cgcgccgtcgggcccggggg gttctctcgg 540 tcacgccgcc cccgacgaag ccgagcgcac ggggtcggcggcgatgtcgg ctacccaccc 600 gacccgtctt gaaacacgga ccaaggagtc taacgcgtgcgcgagtcagg ggctcgcacg 660 aaagccgccg tggcgcaatg aaggtgaagg gccccgtccgggggcccgag gtgggatcc 719 12 685 DNA Mus musculus 12 cgaggcctctccagtccgcc gagggcgcac caccggcccg tctcgcccgc cgcgtcgggg 60 aggtggagcacgagcgtacg cgttaggacc cgaaagatgg tgaactatgc ctgggcaggg 120 cgaagccagaggaaactctg gtggaggtcc gtagcggtcc tgacgtgcaa atcggtcgtc 180 cgacctgggtataggggcga aagactaatc gaaccatcta gtagctggtt ccctccgaag 240 tttccctcaggatagctggc gctctcgcaa ccttcggaag cagttttatc cgggtaaagg 300 cggaatggattaggaggtct tggggccgga aacgatctca aactatttct caaactttaa 360 atgggtaaggaagcccggct cgctggcgtg gagccgggcg tggaatgcga gtgcctagtg 420 ggccacttttggtaagcaga actggcgctg cgggatgaac cgaacgccgg gttaaggcgc 480 ccgatgccgacgctcatcag accccagaaa aggtgttggt tgatatagac agcaggacgg 540 tggccatggaagtcggaatc cgctaaggag tgtgtaacaa ctcacctgcc gaatcaacta 600 gccctgaaaatggatggcgc tggagcgtcg ggcccatacc cggccgtcgc cggcagtcgg 660 aacgggacgggacgggagcg gccgc 685 13 5162 DNA Artificial Sequence Chimeric bacterialplasmid 13 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatctgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgctgagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatgaagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacgcgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcatagcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccgcccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaatagggactttcc 420 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagtacatcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggcccgcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctacgtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtggatagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttgttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacgcaaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaactagagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaagcttggtacc 900 gagctcggat cgatatctgc ggccgcgtcg acggaattca gtggatccactagtaacggc 960 cgccagtgtg ctggaattaa ttcgctgtct gcgagggcca gctgttggggtgagtactcc 1020 ctctcaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaacgaggaggat 1080 ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccatctggtcagaa 1140 aagacaatct ttttgttgtc aagcttgagg tgtggcaggc ttgagatctggccatacact 1200 tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcccaggtccaac 1260 tgcaggtcga gcatgcatct agggcggcca attccgcccc tctccctcccccccccctaa 1320 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctatatgtgattttc 1380 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctgtcttcttgac 1440 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgttgaatgtcgt 1500 gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtagcgaccctttg 1560 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagccacgtgtata 1620 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttggatagttgtgga 1680 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatgcccagaaggt 1740 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacatgtgtttagtc 1800 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcctttgaaaaac 1860 acgatgataa gcttgccaca acccgggatc caccggtcgc caccatggtgagcaagggcg 1920 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgacgtaaacggcc 1980 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaagctgaccctga 2040 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtgaccaccctga 2100 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcacgacttcttca 2160 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaaggacgacggca 2220 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaaccgcatcgagc 2280 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctggagtacaact 2340 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatcaaggtgaact 2400 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccactaccagcaga 2460 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctgagcacccagt 2520 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctggagttcgtga 2580 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggccctagagctc 2640 gctgatcagc ctcgactgtg cctctagttg ccagccatct gttgtttgcccctcccccgt 2700 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaaatgaggaaat 2760 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtggggcaggacag 2820 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgggctctatggc 2880 ttctgaggcg gaaagaacca gctggggctc gagtgcattc tagttgtggtttgtccaaac 2940 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagcttggcgtaat 3000 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattccacacaacatac 3060 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaactcacattaa 3120 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccagctgcattaat 3180 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttccgcttcctcgc 3240 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagctcactcaaagg 3300 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatgtgagcaaaag 3360 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttccataggctcc 3420 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcgaaacccgacag 3480 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctctcctgttccga 3540 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtggcgctttctc 3600 aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaagctgggctgtg 3660 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactatcgtcttgagt 3720 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaacaggattagca 3780 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaactacggctaca 3840 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttcggaaaaagag 3900 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttttttgtttgca 3960 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatcttttctacgg 4020 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatgagattatcaa 4080 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatcaatctaaagta 4140 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggcacctatctcag 4200 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtagataactacga 4260 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagacccacgctcac 4320 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgcagaagtggtc 4380 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagctagagtaagta 4440 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatcgtggtgtcac 4500 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaaggcgagttacat 4560 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatcgttgtcagaa 4620 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataattctcttactg 4680 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaagtcattctgag 4740 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggataataccgcgc 4800 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcggggcgaaaactct 4860 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgcacccaactgat 4920 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacaggaaggcaaaatg 4980 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactcttcctttttc 5040 aatattattg aagcatttat cagggttatt gtctcatgag cggatacatatttgaatgta 5100 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtgccacctgacg 5160 tc 5162

1. A method for detecting or determining delivery and expression ofnucleic acid introduced into a cell comprising; introducing labellednucleic acid molecules that encode a reporter gene into cells; detectinglabelled cells as an indication of delivery of the nucleic acid into acell; and measuring the product of the reporter gene as an indication ofDNA expression in the cell, whereby delivery and expression of nucleicacid molecules in the cell is detected or determined.
 2. The method ofclaim 1, wherein the labelled cells are detected by flow cytometry,fluorimetry, cell imaging or fluorescence spectroscopy.
 3. The method ofclaim 1, wherein the labelled cells are detected by flow cytometry 4.The method of claim 1, wherein the nucleic acid molecule is DNA.
 5. Themethod of claim 1, wherein the label is iododeoxyuridine (IdU or IdUrd)or bromodeoxyuridine (BrdU).
 6. The method of claim 1, wherein thereporter gene encodes a fluorescent protein, or enzyme or antibody. 7.The method of claim 6, wherein the enzyme is a luciferase,β-galactosidase or alkaline phosphatase.
 8. The method of claim 6,wherein the fluorescent protein is a red, green or blue fluorescentprotein.
 9. The method of claim 16, step (a) comprises contacting thenucleic acid molecule is with a delivery agent that comprises a cationiccompound.
 10. The method of claim 9, wherein the compound is selectedfrom the group consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (DOTMA), dioleoylphosphatidylethanolamine(DOPE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propan-aminiumtrifluoroacetate(DOSPA), dioleoyl phosphatidylethanolamine (DOPE), C₅₂H₁₀₆N₆O₄.4CF₃CO₂H,C₈₈H₁₇₈N₈O₄S₂.4CF₃CO₂H, C₄₀H₈₄NO₃P.CF₃CO₂H, C₅₀H₁₀₃N₇O₃.4CF₃CO₂H,C₅₅H₁₁₆N₈O₂.6CF₃CO₂H, C₄₉H₁₀₂N₆O₃.4CF₃CO₂H, C₄₄H₈₉N₅O₃.2CF₃CO₂H,C₄₁H₇₈NO₈P), C₁₀₀H₂₀₆N₁₂O₄S₂.8CF₃CO₂H, C₁₆₂H₃₃₀N₂₂O₉.13CF₃CO₂H,C₄₃H₈₈N₄O₂.2CF₃CO₂H, C₄₃H₈₈N₄O₃.2CF₃CO₂H and(1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride.11. The method of claim 10, wherein the nucleic acid molecules arenatural chromosomes, artificial chromosomes, fragments of a chromosomeor naked DNA that is greater than at least about 0.6 megabase in size.12. The method of claim 1, wherein the nucleic acid molecules areartificial chromosomes, plasmids, chromosome fragments, naked DNA, ornatural chromosomes.
 13. The method of claim 1, wherein the nucleic acidmolecules are artificial chromosome expression systems (Aces).
 14. Themethod of claim 1, wherein the cells are eukaryotic cells.
 15. Themethod of claim 14, wherein the cells are primary cells, cell lines,plant cells, animal cells.
 16. The method of claim 15, wherein thecells, are stem cells, nuclear transfer donor cells, tumor cells ortransformed cells.
 17. A method for monitoring the delivery of a nucleicacid molecule into a cell comprising: (a) labeling the nucleic acidmolecule; (b) delivering labeled nucleic acid molecule into a cell; and(c) detecting labeled nucleic acid molecule in the cells by flowcytometry, fluorimetry, cell imaging or fluorescence spectroscopy, as anindication of delivery of nucleic acid molecule into the cells.
 18. Themethod of claim 17, wherein the nucleic acid molecule is labeled with athymidine analog.
 19. The method of claim 18, wherein the thymidineanalog is iododeoxyuridine or bromodeoxyuridine.
 20. The method of claim19, wherein a delivery agent comprises a cationic compound, and thenucleic acid molecule is treated therewith.
 21. The method of claim 20,wherein the compound is selected from the group consisting ofN-[1-(2,3-dioleyloxy)propyl]-N,N,N -trimethyl-ammonium chloride (DOTMA),dioleoylphosphatidylethanolamine (DOPE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propan-aminiumtrifluoroacetate(DOSPA), dioleoyl phosphatidylethanolamine (DOPE), C₅₂H₁₀₆N₆O₄.4CF₃CO₂H,C₈₈H₁₇₈N₈O₄S₂.4CF₃CO₂H, C₄₀H₈₄NO₃P.CF₃CO₂H, C₅₀H₁₀₃N₇O₃.4CF₃CO₂H,C₅₅H₁₁₆N₈O₂.6CF₃CO₂H, C₄₉H₁₀₂N₆O₃.4CF₃CO₂H, C₄₄H₈₉N₅O₃.2CF₃CO₂H,C₄₁H₇₈NO₈P), C₁₀₀H₂₀₆N₁₂O₄S₂.8CF₃CO₂H, C₁₆₂H₃₃₀N₂₂O₉.13CF₃CO₂H,C₄₃H₈₈N₄O₂.2CF₃CO₂H, C₄₃H₈₈N₄O₃.2CF₃CO₂H and(1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride.22. The method of claim 18, wherein the nucleic acid molecule is anatural chromosome, an artificial chromosome, a fragment of a chromosomeor naked DNA that is greater than at least about 0.6 megabase in size.23. A method for screening agents for the ability to deliver nucleicacid molecule into a cell comprising: (a) delivering a labeled nucleicacid molecule into the cell in the presence of the agent; and (b)determine the number of cells containing the label, as an indication ofthe ability of the agent to deliver nucleic acid molecule into the cell.24. The method of claim 23, wherein the number of cells is determined byflow cytometry, fluorimetry, cell imaging or fluorescence spectroscopy.25. The method of claim 23, wherein the label is iododeoxyuridine orbromodeoxyuridine.
 26. The method of claim 23, wherein the agentcomprises a cationic compound.
 27. The method of claim 26, wherein thecompound is selected from the group consisting ofN-[1-(2,3-dioleyloxy)propyl]-N,N,N -trimethyl-ammonium chloride (DOTMA),dioleoylphosphatidylethanolamine (DOPE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propan-aminiumtrifluoroacetate(DOSPA), dioleoyl phosphatidylethanolamine (DOPE), C₅₂H₁₀₆N₆O₄.4CF₃CO₂H,C₈₈H₁₇₈N₈O₄S₂.4CF₃CO₂H, C₄₀H₈₄NO₃P.CF₃CO₂H, C₅₀H₁₀₃N₇O₃.4CF₃CO₂H,C₅₅H₁₁₆N₈O₂.6CF₃CO₂H, C₄₉H₁₀₂N₆O₃.4CF₃CO₂H, C₄₄H₈₉N₅O₃.2CF₃CO₂H,C₄₁H₇₈NO₈P), C₁₀₀H₂₀₆N₁₂O₄S₂.8CF₃CO₂H, C₁₆₂H₃₃₀N₂₂O₉.13CF₃CO₂H,C₄₃H₈₈N₄O₂.2CF₃CO₂H C₄₃H₈₈N₄O₃.2CF₃CO₂H and(1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride.28. The method of claim 23, wherein the nucleic acid molecule is anatural chromosome, an artificial chromosome, a fragment of a chromosomeor naked DNA, or a plasmid
 29. The method of claim 23, wherein thenucleic acid molecule is a natural chromosome, an artificial chromosome,a fragment of a chromosome or naked DNA that is greater than at leastabout 0.6 megabase in size.
 30. The method of claim 1, wherein the cellis selected from the group consisting of a primary cell, an immortalizedcell, an embryonic cell, a stem cell, a transformed cells and a tumorcell.
 31. The method of claim 17, wherein the cell is selected from thegroup consisting of a primary cell, an immortalized cell, an embryoniccell, a stem cell, a transformed cells and a tumor cell.
 32. The methodof claim 23, wherein the cell is selected from the group consisting of aprimary cell, an immortalized cell, an embryonic cell, a stem cell, atransformed cells and a tumor cell.